Download Cephalostatin 1 Selectively Triggers the Release

[CANCER RESEARCH 63, 8869–8876, December 15, 2003]
Cephalostatin 1 Selectively Triggers the Release of Smac/DIABLO and Subsequent
Apoptosis That Is Characterized by an Increased Density of the
Mitochondrial Matrix
Verena M. Dirsch,1 Irina M. Müller,1 Sören T. Eichhorst,2 George R. Pettit,4 Yoshiaki Kamano,4 Masuo Inoue,4
Jun-Ping Xu,4 Yoshitatsu Ichihara,4 Gerhard Wanner,3 and Angelika M. Vollmar1
1
Department of Pharmacy, Center of Drug Research, 2Department of Medicine II, Klinikum Grosshadern, and 3Department of Biology I, University of Munich, Munich, Germany,
and 4Cancer Research Institute, Arizona State University, Tempe, Arizona
ABSTRACT
Cephalostatin 1 is a bis-steroidal marine natural product with a unique
cytotoxicity profile in the in vitro screen system of the National Cancer
Institute, suggesting that it may affect novel molecular target(s). Here we
show that cephalostatin 1 induces a novel pathway of receptor-independent apoptosis that selectively uses Smac/DIABLO (second mitochondriaderived activator of caspases/direct inhibitor of apoptosis-binding protein
with a low isoelectric point) as a mitochondrial signaling molecule. At
nanomolar concentrations, cephalostatin 1 triggers dose- and time-dependent DNA fragmentation in leukemia Jurkat T cells. Apoptosis was found
to be dependent on caspase activity because the pan-caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone blocks cephalostatin 1-mediated DNA fragmentation. The CD95 death receptor as well
as other caspase-8-requiring death receptors were not involved because
Jurkat T cells lacking the CD95 receptor or caspase-8 and control cells
responded equally to cephalostatin 1. Although cephalostatin 1 affects
mitochondria by dissipating the mitochondrial membrane potential, neither cytochrome c nor apoptosis-inducing factor is released, as shown by
Western blot analysis. Interestingly, cephalostatin 1 selectively triggers
the mitochondrial release of the inhibitor of apoptosis antagonist Smac/
DIABLO. Overexpression of the antiapoptotic protein Bcl-xL delayed both
Smac/DIABLO release and onset of apoptosis, suggesting that Smac/
DIABLO is required for cephalostatin 1-induced apoptosis. This new
mitochondrial pathway is accompanied by marked structural changes of
mitochondria as shown by transmission electron microscopy.
INTRODUCTION
Cephalostatins are remarkable antineoplastic bis-steroidal natural
products showing activity in the subnanomolar to nanomolar range.
Cephalostatin 1, the first member of this series of compounds (cephalostatin 1–19), was detected in 1972 and subsequently isolated from
extracts of Cephalodiscus gilchristi, a small Southeast African marine
worm. The structure was reported in 1988 (1). In the 60-cell line in
vitro screen of the National Cancer Institute, cephalostatin 1 yielded
a mean panel 50% growth inhibition concentration of approximately
1 nM (2). Cephalostatin 1 is now in preclinical development. However,
the understanding of the antineoplastic activity of cephalostatin 1 at
the molecular level has remained elusive.
A wide range of chemotherapeutic drugs induce death in malignant
cells by triggering apoptosis (3). Two major apoptotic pathways used
by cytotoxic drugs have been described: one that begins with the
ligation of death receptors and subsequently activates the initiator
caspase-8; and another that integrates various cell stress signals via
the “activation” of mitochondria. One crucial event in the mitochondrial pathway is the release of cytochrome c from the mitochondria
into the cytosol. Once released, cytochrome c initiates the assembly of
apoptotic protease-activating factor 1 (Apaf-1) and procaspase-9 into
a holoenzyme complex called the “apoptosome,” which in turn activates the initiator caspase-9 (4). Initially activated caspase-8 (receptor-mediated pathway; extrinsic pathway) or caspase-9 (mitochondrial
pathway; intrinsic pathway) activates downstream caspases, such as
caspase-3, the activation of which results in DNA fragmentation and
apoptosis.
A current view of drug-induced apoptosis emphasizes the role of
different cell organelles [e.g., mitochondria, cytoskeleton, nucleus,
plasma membrane, lysosomes, and the endoplasmic reticulum (ER)]
as stress sensors that either reroute the signal directly through mitochondria or in some cases activate their own death pathways (5, 6).
The central role of mitochondria in drug-induced apoptosis implies
that alterations in this particular pathway play an important role in
drug resistance. Indeed, resistance to chemotherapy was found to be
related to increased levels of the mitochondria-protecting proteins
Bcl-2 and Bcl-xL (4, 7, 8). Besides these, inhibitors of apoptosis
proteins (IAPs) are shown to be overexpressed in cancer and are
associated with resistance to apoptosis (7, 9). Some IAP family
proteins bind and inhibit the activation of the initiator caspase-9 as
well as activated effector caspases-3 and -7 (4, 9) and are thus
endogenous repressors of the mitochondrial pathway of apoptosis.
IAPs are antagonized by Smac/DIABLO (second mitochondriaderived activator of caspases/direct IAP-binding protein with a low
isoelectric point). Mature Smac/DIABLO is a homodimer of two
21-kDa triple ␣-helical bundle monomers that reside in the mitochondrial intermembrane space. It is released during apoptosis into the
cytosol, where it can interact with IAPs, thereby promoting caspase
activation (10).
New drugs that induce apoptosis by mechanisms differing from
those of classical chemotherapeutic drugs might provide a chance to
overcome chemoresistance. The unique differential cytotoxicity profile of cephalostatins obtained from the 60-cell line in vitro screen of
the National Cancer Institute provides a good possibility that cephalostatins may affect novel molecular targets (2). Therefore, the aims of
the present study were (a) to examine whether cephalostatin 1 induces
apoptosis in leukemia Jurkat T cells and (b) to focus on the characterization of the apoptotic pathways involved.
MATERIALS AND METHODS
Received 9/17/02; revised 9/10/03; accepted 10/15/03.
Grant support: Supported by Deutsche Forschungsgemeinschaft Grant SFB 369 and
Outstanding Investigator Grants CA 44344-1-12 and ROI CA90441-1-2 from the Division
of Cancer Treatment and Diagnosis, National Cancer Institute, Department of Health and
Human Services.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Verena M. Dirsch, Department of Pharmacy, Center of Drug
Research, Butenandtstrasse, 5-13, D-81377 Munich, Germany. Phone: 49-89-2180-77161;
Fax: 49-89-2180-77173; E-mail: [email protected].
Compounds. Cephalostatin 1 was isolated from C. gilchristi (order,
Cephalodiscida) as described previously (1). Purity of the compound was at
least 98% as judged by high-performance liquid chromatography analysis.
Before application, cephalostatin 1 was dissolved and further diluted in
DMSO. Final DMSO concentration did not exceed 1%, a concentration verified not to interfere with the experiments performed. Etoposide, staurosporine,
and the caspase inhibitors were purchased from Calbiochem (Bad Soden,
Germany). The soluble CD95 ligand was from Alexis Biochemicals (Grün8869
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CEPHALOSTATIN 1-INDUCED APOPTOSIS
berg, Germany), and propidium iodide (PI) was obtained from Sigma (Deisenhofen, Germany).
Cell Culture. Human leukemia Jurkat T cells (clone J16), the CD95resistant JurkatR (11), Jurkat T cells transfected with vector control or Bcl-xL
(12), Jurkat T cells (clone A3), and caspase-8-deficient A3 [Ref. 13; kindly
provided by Drs. P. H. Krammer and H. Walczak (Heidelberg, Germany) and
Dr. K. Schulze-Osthoff (Münster, Germany)] were cultured (37°C and 5%
CO2) in RPMI 1640 containing 2 mM L-glutamine (PAN Biotech, Aidenbach,
Germany) supplemented with 10% FCS (PAA Laboratories, Cölbe, Germany).
Medium of transfected cells was supplemented with 1 mg/ml G418 (Life
Technologies, Inc., Eggenstein, Germany) every fifth passage.
Cell Viability. Impaired cell viability was measured using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, based on the
ability of viable cells to reduce yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to blue formazan, as described previously (14).
Detection and Quantification of Apoptosis. Apoptosis was judged by (a)
the visualization of apoptotic nuclei after staining with Hoechst 33342 (Sigma)
by fluorescence microscopy and (b) translocation of phosphatidylserine to the
cell surface using an annexin V-FITC apoptosis detection kit (Calbiochem).
Quantification of apoptosis was performed as described by Nicoletti et al. (15).
Briefly, cells were incubated for 24 h in a hypotonic buffer (0.1% sodium
citrate, 0.1% Triton X-100, and 50 ␮g/ml PI) and analyzed by flow cytometry
on a FACSCalibur (Becton Dickinson, Heidelberg, Germany). Nuclei to the
left of the G1 peak containing hypoploid DNA were considered apoptotic.
Analysis of Mitochondrial Transmembrane Potential (⌬␺m). Cells
were stained with the fluorochrome 5,5⬘,6-6⬘tetrachloro-1,1⬘,3,3⬘-tetraethylbenzimidazolcarbo-cyanine iodide [JC-1 (1.25 ␮g/ml); Molecular Probes, Eugene, OR] as described by Bernardi et al. (16) and Cossarizza et al. (17). The
membrane potential was measured by fluorescence-activated cell-sorting
(FACS) analysis. JC-1 aggregates were detected at 585 nm (FL-2), and JC-1
monomers were detected at 530 nm (FL-1). The percentage of cells with pure
green fluorescence was calculated by gate analysis on a FACSCalibur using
CellQuest software (Becton Dickinson).
Measurement of Cytochrome c Release. Release of cytochrome c from
mitochondria was analyzed as described by Leist et al. (18). Briefly, cell
pellets were resuspended in permeabilization buffer [210 mM D-mannitol, 70
mM sucrose, 10 mM HEPES, 5 mM succinate, 0.2 mM EGTA, 0.15% BSA, and
60 ␮g/ml digitonin (pH 7.2), 4°C] and incubated for 20 min at 4°C. Permeabilized cells were centrifuged (300 ⫻ g), and the supernatant was removed
and centrifuged again (10 min; 13,000 ⫻ g). The obtained cytosol was
separated by 15% SDS-PAGE and probed for cytochrome c as described
below. The remaining pellet of permeabilized cells was lysed in 0.1% Triton/
PBS (15 min; 4°C) and centrifuged (12,000 ⫻ g; 4°C; 10 min), and the
supernatant containing mitochondrial cytochrome c was analyzed by
SDS-PAGE.
Western Blot Analysis and Immunoprecipitation. Cells were collected
by centrifugation, washed with ice-cold PBS, and lysed in 1% Triton X-100,
0.15 M NaCl, and 30 mM Tris-HCl (pH 7.5) with the protease inhibitor
Complete (Roche, Mannheim, Germany) for 30 min. Lysates were homogenized through a 22-gauge needle and centrifuged at 10,000 ⫻ g for 10 min at
4°C. Equal amounts of protein were separated by SDS-PAGE [7.5% SDSPAGE for Apaf-1 and poly(ADP-ribose) polymerase, 10% SDS-PAGE for
caspase-8 and caspase-9, 12% SDS-PAGE for caspase-3 and X-linked IAP
(XIAP), 15% SDS-PAGE for cytochrome c, apoptosis-inducing factor (AIF),
and Smac/DIABLO] and transferred to polyvinylidene difluoride membranes
(Immobilon-P; Millipore, Eschborn, Germany). Equal protein loading was
controlled by Coomassie Blue staining of gels. Membranes were blocked with
5% fat-free milk powder in PBS containing 0.05% Tween 20 (1 h) and
incubated with specific antibodies against caspase-3 (mouse IgG2a; BD Biosciences, Heidelberg, Germany), caspase-8 (mouse monoclonal antibody,
clone 5F7; Upstate Biotechnology, Lake Placid, NY), caspase-9 (mouse monoclonal IgG1, clone Ab-2; Oncogene, San Diego, CA), Apaf-1 (mouse IgG2a;
BD Biosciences), XIAP (rabbit polyclonal antibody; Chemicon, Temecula,
CA), poly(ADP-ribose) polymerase (mouse IgG1; Oncogene), cytochrome c
(mouse monoclonal antibody 7H8.2C12; BD Biosciences), AIF (rabbit polyclonal antibody; Upstate Biotechnology), and Smac/DIABLO (Serotec, Oxford, United Kingdom) overnight at 4°C. Specific proteins were visualized by
secondary antibodies conjugated to horseradish peroxidase and the Renais-
sance Plus reagent (NEN Life Science, Zaventem, Belgium). Pictures were
taken on a Kodak Digital Science Image station 440CF (NEN Life Science).
For immunoprecipitation, cells were lysed as described previously. Lysates
were cleared by centrifugation, and protein concentration was determined.
Anti-Smac antibody and anti-caspase-9 antibody (2.5 ␮l), respectively, were
added to 250 ␮g of protein (1 ␮g/␮l) and mixed overnight at 4°C. Washed
protein A-agarose beads (50 ␮l) were added for an additional 2–3 h and
subsequently collected by centrifugation. The beads were washed three times
with 500 ␮l of lysis buffer and resuspended in 25 ␮l of a 3⫻ sample buffer
containing 1.5% ␤-mercaptoethanol. After addition of 25 ␮l of 1⫻ sample
buffer, beads were boiled for 5 min at 95°C and then pelleted by short spin.
Thirty ␮l of the supernatant were used for SDS-PAGE.
Analysis of Caspase-3 Activity. Cells were treated with 1 ␮M cephalostatin 1 for the indicated times. Etoposide (25 ␮g/ml) was used as positive
control. Stimulated cells were collected by centrifugation, washed twice with
ice-cold PBS, resuspended in lysis buffer [5 mM MgCl2, 1 mM EGTA, 0.1%
Triton X-100, and 25 mM HEPES (pH 7.5)] and kept overnight at ⫺80°C.
After defrosting, lysates were cleared by centrifugation. Substrate solution
containing 55 ␮M benzyloxycarbonyl-Asp-Glu-Val-Asp aminofluoromethylcoumarin was added to the supernatant, and generation of free 7-amino-4trifluoromethyl coumarin at 37°C was determined by fluorescence measurement in a SpectraFluor Plus plate reader (Tecan) at an excitation wavelength
of 390 nm and an emission wavelength of 535 nm.
Confocal Laser Scanning Microscopy. Cells were stimulated with cephalostatin 1 (1 ␮M) or etoposide (25 ␮g/ml) for 8 h. Cells (4 ⫻ 104) were
centrifuged onto glass slides and dried at room temperature overnight. Samples
were fixed and permeabilized with 4% paraformaldehyde containing 0.05%
saponin in PBS for 15 min at room temperature. After washing with 0.03%
saponin in PBS, probes were blocked with 1% BSA, 1% FCS, and 0.1% Tween
20 in PBS for 1 h and washed twice in PBS. Anti-cytochrome c antibody and
anti-Smac antibody were applied together (1:100 dilution in blocking buffer)
for costaining. Anti-AIF was used at the same concentration. As secondary
antibodies, Alexa Fluor 488 antimouse IgG antibody, antirabbit antibody, and
Alexa Fluor 633 antimouse IgG antibody (Molecular Probes) were used at a
dilution of 1:200. Pictures were taken by Zeiss Meta confocal laser scanning
microscopy.
Transmission Electron Microscopy. Cells were collected by centrifugation, washed once in PBS, resuspended, and fixed in fixing solution [2.5%
glutaraldehyde in the following fixative buffer: 75 mM cacodylate, 75 mM
NaCl, and 2 mM MgCl2 (pH 7.0)] for 1 h. Subsequently, cells were washed
several times for increasing periods in fixative buffer, postfixed in 1% OsO4 in
fixative buffer, and washed with buffer and Aqua dest. Cells were dehydrated
with a graded series of acetone. Cells were infiltrated with Spurr low-viscosity
epoxyresin and polymerized at 65°C. Pictures were taken with a LEO EM 912
transmission electron microscope with integrated ⍀-filter operated in “zeroloss” mode.
Statistical Analysis. All experiments were performed at least three times in
triplicate. Results are expressed as mean value ⫾ SE. One-way ANOVA with
Dunnett’s post test and Student’s unpaired two-tailed t test were performed
using GraphPad Prism version 3.0 for Windows (GraphPad Software, San
Diego, CA). P ⬍ 0.05 was considered significant.
RESULTS
Cephalostatin 1 Induces Apoptosis in Jurkat Leukemia T Cells.
Initial dose-response studies using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay revealed that 1 ␮M cephalostatin 1 effectively impairs cell viability of Jurkat T cells (IC50 ⫽ 1.25
nM). This cytotoxicity was attributable to the induction of apoptosis as
shown by the following morphological and biochemical features: 1
␮M cephalostatin 1 led to the characteristic apoptotic morphology with
shrunken cells and apoptotic bodies (data not shown). Nuclei fragmentation was detected by fluorescence microscopy after DNA staining with Hoechst 33342 (Fig. 1A, left panel) and by the appearance of
nuclei with subdiploid DNA content as shown by flow cytometry after
PI staining (Fig. 1A, middle panel). Annexin V-FITC labeling revealed the exposure of phosphatidylserine on the outside of the
plasma membrane (Fig. 1A, right panel).
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16 h after stimulation. In contrast, the processing of the proform of
caspase-9 to the active forms (p35/p37) occurred after only 4 h (Fig.
2C). To test whether caspase-3, an important effector caspase, is
activated downstream of caspase-9, the processing of caspase-3 (Fig.
2D) and poly(ADP-ribose) polymerase cleavage (Fig. 2E) in response
to cephalostatin 1 (1 ␮M) was demonstrated.
The CD95 Receptor and Caspase-8 Are Not Required for Apoptosis Induction by Cephalostatin 1. The extrinsic pathway involves death receptors such as the CD95 receptor, the activation of
which processes caspase-8. To study whether the CD95 receptor is
participating in cephalostatin 1-induced apoptosis, we treated control
cells and CD95 receptor-deficient JurkatR cells (11) with cephalostatin 1 (0.1–1 ␮M; 24 h). In JurkatR cells, no reduction of apoptosis
could be detected compared with control cells (Fig. 3A). Thus, the
CD95 receptor is not required for cephalostatin-induced apoptosis.
Because other death receptors also use caspase-8 as an initiator
caspase, we examined whether caspase-8 activation is necessary for
apoptosis or whether caspase-8 is merely activated downstream as a
consequence of a caspase amplification loop in response to cephalostatin 1. Fig. 3B shows that control cells and cells deficient in
caspase-8 (caspase-8⫺/⫺ cells) respond equally to cephalostatin 1
Fig. 1. Cephalostatin 1 induces apoptosis in Jurkat leukemia T cells. A, left panel, cells
were treated with cephalostatin 1 (1 ␮M; 16 h) and subsequently stained with Hoechst
33342. Representative pictures of untreated (Control) and cephalostatin 1-treated cells are
shown. Middle panel, FACS analysis of PI-stained nuclei of cells treated as described
above for 24 h. Histograms show the distribution of nuclei according to their DNA
content. Counts left of the G1-peak (gated) demonstrate the appearance of nuclei with
subdiploid DNA content. Right panel, FACS analysis of annexin V-FITC and PI-stained
cells, either left untreated (Control) or stimulated with cephalostatin 1 (1 ␮M, 8 h). Cells
appearing in the bottom right quadrant show positive annexin V-FITC staining that
indicates phospatidylserine exposure on the cell surface and no DNA staining with PI,
proving intact cell membranes. B, cephalostatin 1-induced apoptosis is time and dose
dependent. Cells were stimulated with increasing concentrations of cephalostatin 1 for
24 h (top panel) or cephalostatin 1 (1 ␮M) for the indicated periods of time (bottom panel).
% Apoptotic cells, percentage of cells with subdiploid DNA content as described in
“Materials and Methods.” Bars, mean ⫾ SE of three independent experiments performed
in triplicate. ⴱⴱ, P ⬍ 0.001 (ANOVA/Dunnett’s test).
Apoptosis induction by cephalostatin 1 occurred in a dose- and
time-dependent manner. The appearance of apoptotic cells was significant 6 h after exposure to cephalostatin 1 (1 ␮M) and at a concentration as low as 1 nM (24 h; Fig. 1B).
Cephalostatin 1-Induced Apoptosis Is Dependent on the Activation of Caspases. To elucidate whether caspase activation is required for cephalostatin 1-induced apoptosis, cells were preincubated
with the broad-spectrum caspase inhibitor benzyloxycarbonyl-ValAla-Asp(OMe)-fluoromethylketone (zVAD-fmk). Caspase inhibition
led to a complete inhibition of cephalostatin 1-induced DNA fragmentation (Fig. 2A), proving the importance of caspases in this
apoptotic process. To get a better idea of whether cephalostatin
1-triggered apoptosis follows the extrinsic pathway including activation of the initiator caspase-8 or the intrinsic pathway under involvement of mitochondria and the initiator caspase-9, we examined the
activation of these caspases by Western blot analysis. Fig. 2B illustrates the time-dependent activation of caspase-8 by cephalostatin 1 (1
␮M). The caspase-8 cleavage product p42/p44 did not appear until
Fig. 2. Cephalostatin 1-induced apoptosis is dependent on the activation of caspases.
A, inhibition of cephalostatin 1-induced apoptosis by the caspase inhibitor zVAD-fmk.
Cells were left untreated (Control), incubated with cephalostatin 1 (1 ␮M, 24 h), or
pretreated with zVAD-fmk (25 ␮M, 1 h) and then stimulated with cephalostatin 1 (1 ␮M,
24 h). Etoposide (E; 25 ␮g/ml, 24 h) was used as positive control. Apoptotic cells were
quantified by flow cytometry as described in “Materials and Methods.” B⫺E, representative Western blots showing time-dependent (0 –24 h) cleavage as follows: B,
procaspase-8 isoforms are cleaved to the p42 and p44 cleavage products; C, procaspase-9
is cleaved to the active cleaved forms p35 and p37; D, procaspase-3 is cleaved to p17; and
E, 116-kDa PARP is cleaved to its p89 cleavage product after treatment with cephalostatin
1 (1 ␮M). Cells were lysed, and proteins were separated by SDS-PAGE as described in
“Materials and Methods.” All experiments were performed three times with consistent
results. Bars, mean ⫾ SE of three independent experiments performed in triplicate; ⴱⴱ,
P ⬍ 0.001 (ANOVA/Dunnett’s test). PARP, poly(ADP-ribose) polymerase.
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Fig. 3. Cephalostatin 1 induces apoptosis independently of the CD95 receptor and
caspase-8. A, Jurkat and JurkatR cells were treated with the indicated concentrations of
cephalostatin 1 for 24 h. B, Jurkat A3 and Jurkat caspase-8⫺/⫺ cells were stimulated with
cephalostatin 1 (1 ␮M) for different time periods (0 –24 h). C, Jurkat A3 (left panel) and
Jurkat caspase-8⫺/⫺ cells (right panel) were either left untreated (Co) or treated with
soluble CD95 ligand (200 ng/ml) or etoposide (E, 25 ␮g/ml). A⫺C, apoptotic cells were
quantified by FACS analysis as described in “Materials and Methods.” All experiments
were carried out three times in triplicate. Data points and bars, the mean ⫾ SE of three
independent experiments; ⴱⴱ, P ⬍ 0.001; n.s., not significant (ANOVA/Dunnett’s test).
CD95-L, CD95 ligand.
(1 ␮M), suggesting that activation of caspase-8 is not required for
cephalostatin 1-induced apoptosis. To verify that the used cell line
does not express caspase-8, we performed Western blot analysis (data
not shown) and treated Jurkat A3 (control cells) and Jurkat caspase8⫺/⫺ cells with soluble CD95 ligand or etoposide (25 ␮g/ml). As
expected, Jurkat caspase-8⫺/⫺ cells were protected against CD95
ligand but not against etoposide (Fig. 3C). These results, together with
the fact that caspase-8 succeeds caspase-9 activation, clearly show
that cephalostatin 1 does not induce apoptosis via the extrinsic pathway.
Cephalostatin 1 Induces Inner Mitochondrial Membrane Permeabilization and Release of Smac/DIABLO but not Release of
Cytochrome c or AIF. The observed early activation of the initiator
caspase-9 in response to cephalostatin 1 (Fig. 2) points to a putative
mitochondria-mediated signaling. Hallmarks of the mitochondrial
pathway of apoptosis (intrinsic pathway) are the release of cytochrome c from the mitochondrial intermembrane space into the cytosol and the dissipation of the electrochemical gradient (⌬␺m) on the
inner mitochondrial membrane (inner mitochondrial membrane permeabilization). Fig. 4A shows that cephalostatin 1 (1 ␮M) induces
inner mitochondrial membrane permeabilization in Jurkat T cells as
measured by flow cytometry using the fluorochrome JC-1. The first
Fig. 4. Cephalostatin 1 induces inner mitochondrial membrane permeabilization and
the mitochondrial release of Smac/DIABLO but not the release of cytochrome c or AIF.
A, cephalostatin 1 leads to a time-dependent dissipation of ⌬␺m. Jurkat cells were
incubated with cephalostatin 1 (1 ␮M) for the indicated time periods and subsequently
loaded with the fluorochrome JC-1 (0.25 ␮g/ml). Cells showing predominantly green
fluorescence (low ⌬␺m) were quantified by FACS analysis as described in “Materials and
Methods.” Data points, the mean ⫾ SE of three independent experiments performed in
triplicate. ⴱ, P ⬍ 0.05; ⴱⴱⴱ, P ⬍ 0.001 (ANOVA/Dnnett’s test). B and C, cephalostatin
1 does not lead to cytochrome c (B) or AIF (C) release into the cytosol. Cells were treated
with cephalostatin 1 (1 ␮M) for the indicated times or with etoposide (E; 25 ␮g/ml, 16 h)
as a positive control. Cytosol and mitochondrial protein were prepared as described in
“Materials and Methods” and detected by specific antibodies using Western blot analysis.
D, cells were pretreated with zVAD-fmk (25 ␮M; 1 h) where indicated and subsequently
stimulated with cephalostatin 1 (1 ␮M) for 2 and 4 h. Cytosol and mitochondrial protein
were prepared and analyzed as described in B and C. Representative Western blots are
shown. E, cells were either left untreated (CO) or stimulated with cephalostatin 1 (CPH;
1 ␮M) or etoposide (ETO; 25 ␮g/ml) for 8 h and prepared for confocal laser scanning
microscopy as described in “Materials and Methods.” Representative pictures are shown.
Cyt-c, cytochrome c.
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significant dissipation of ⌬␺m was evident 8 h after stimulation. Most
surprisingly, cephalostatin 1 (1 ␮M; 0 –24 h) does not trigger the
release of cytochrome c into the cytosol. Etoposide (25 ␮g/ml) was
used as positive control (Fig. 4B). Thus, cephalostatin 1 activates
caspase-9 independently of cytochrome c.
Besides cytochrome c, additional proapoptotic factors present
within mitochondria and released after various apoptotic stimuli are
the AIF and Smac/DIABLO (4). Fig. 4C demonstrates that similar to
cytochrome c, AIF is not released in response to cephalostatin 1. In
striking contrast, cephalostatin 1 triggers an early (2 h) and intense
release of Smac/DIABLO that is not abrogated by the pan-caspase
inhibitor zVAD-fmk. This indicates that cephalostatin 1-induced
Smac/DIABLO release is an early event that occurs before and
independent of caspase activation.
This unique property (i.e., the selective release of Smac/DIABLO)
of cephalostatin 1 was confirmed by immunofluorescence experiments detecting cytochrome c, Smac/DIABLO, and AIF in response
to cephalostatin (1 ␮M) and etoposide (25 ␮g/ml) by confocal laser
scanning microscopy. Fig. 4E demonstrates that in untreated Jurkat
cells, cytochrome c and Smac/DIABLO colocalize at distinct areas
(mitochondria). In response to cephalostatin, Smac/DIABLO is released, whereas cytochrome c still displays a punctuate mitochondrial
appearance. In contrast, etoposide leads to a diffuse pattern of both
cytochrome c and Smac/DIABLO. Similar to Western blot analysis
(Fig. 4C), AIF translocation was not detected and was hardly detected
in response to cephalostatin and etoposide, respectively.
Overexpression of Bcl-xL Delays Smac/DIABLO Release and
Onset of Cephalostatin-Induced Apoptosis. To clarify whether
Smac/DIABLO is required for cephalostatin 1-triggered apoptosis, we
examined whether overexpression of Bcl-xL, a mitochondria-protecting protein that is shown to prevent the release of Smac/DIABLO
(19), also inhibits apoptosis. Initially, Jurkat/neo and Jurkat/bcl-xL
cells were verified to differ in Bcl-xL expression by Western blot
analysis (data not shown) and by their response to staurosporine [500
nM, 24 h (20, 21)]. As seen in Fig. 5A, staurosporine readily kills
Jurkat/neo cells, whereas Jurkat/bcl-xL cells are protected against
staurosporine. Both cell types were then exposed to cephalostatin 1 (1
␮M) for 0 –24 h. Fig. 5B shows that Smac/DIABLO release is significantly delayed in Jurkat/bcl-xL cells compared with Jurkat/neo cells
(2 versus 16 h). Corresponding to this delayed Smac/DIABLO release, onset of DNA fragmentation in response to cephalostatin 1 is
delayed (8 h for Jurkat/neo versus 24 h for Jurkat/bcl-xL; Fig. 5C).
Interestingly, dissipation of ⌬␺m was not affected by overexpression
of Bcl-xL (Fig. 5D). This observation, together with its late appearance (16 h), indicates that dissipation of ⌬␺m is a secondary event
within the cephalostatin 1-induced signaling cascade.
Caspase-9 Is Not Recruited into an Apaf-1-Containing Apoptosome and Does Not Act as a Major Initiator Caspase Activated
in Response to Cephalostatin 1. Next, we sought to clarify whether,
in the absence of cytochrome c, cephalostatin 1-activated caspase-9 still
acts as essential initiator caspase. Therefore, we examined whether
caspase-9 is recruited into the apoptosome by binding to Apaf-1 in
cephalostatin 1-activated cells. Western blot analysis of caspase-9 immunoprecipitates (Fig. 6A) shows that in etoposide (25 ␮g/ml)-activated
cells, caspase-9 associates with Apaf-1, whereas cephalostatin (1 ␮M)
treatment does not lead to caspase-9-Apaf-1 association. This suggests
that cephalostatin 1 treatment does not induce the formation of an
apoptosome. This outcome posed the question of whether caspase-9 is
still essential for cephalostatin 1-mediated apoptosis. Inhibition of
caspase-9 by the caspase-9 inhibitor benzyloxycarbonyl-Leu-Glu(OMe)His-Asp(OMe)-fluoromethylketone (50 ␮M) led to an inhibition of
etoposide-induced caspase-3 activity (Fig. 6B) but not to a complete
inhibition of cephalostatin 1-induced apoptosis (Fig. 6C). This result
Fig. 5. Bcl-xL delays mitochondrial Smac/DIABLO release and onset of apoptosis but not
dissipation of ⌬␺m. A, control cells (Jurkat/neo) and cells overexpressing Bcl-xL (Jurkat/bclxL) were incubated with staurosporine (500 nM, 24 h). Apoptotic cells were quantified by flow
cytometry as described in “Materials and Methods.” B, Jurkat/neo (top panels) and L Jurkat/
bcl-xL, (bottom panels) cells were treated with cephalostatin 1 (1 ␮M) for the indicated times
or with etoposide (E; 25 ␮g/ml, 16 h) as a positive control. Cytosol and mitochondrial proteins
were prepared as described in “Materials and Methods,” and Smac/DIABLO was detected by
a specific antibody using Western blot analysis. A representative Western blot is shown. C,
Jurkat/neo and Jurkat/bcl-xL cells were incubated with cephalostatin 1 (1 ␮M) for the indicated
times. Apoptosis was quantified by flow cytometry as described in A. D, cells treated as
described in B were loaded with the fluorochrome JC-1 (0.25 ␮g/ml). Cells showing predominantly green fluorescence (low ⌬␺m) were quantified by FACS analysis as described in
“Materials and Methods.” Data points and bars, the mean ⫾ SE of three independent
experiments performed in triplicate. ⴱ, P ⬍ 0.05; ⴱⴱⴱ, P ⬍ 0.001 (A, unpaired two-tailed t test;
C and D, ANOVA/Dunnett’s test).
indicates that cephalostatin 1-activated caspase-9 contributes to but is
not essential for cephalostatin 1-mediated apoptosis.
Cephalostatin 1-Released Smac/DIABLO Displaces XIAP from
Caspase-9. Smac/DIABLO is suggested to act as an apoptosis promoter by interacting with IAPs, preventing these proteins from bind-
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CEPHALOSTATIN 1-INDUCED APOPTOSIS
Fig. 6. A, cephalostatin 1 treatment does not lead to association of caspase-9 with
Apaf-1. Cells were either left untreated (CO) or stimulated with cephalostatin 1 (CPH; 1
␮M) or etoposide (ETO; 25 ␮g/ml) for the indicated time, and lysates were immunoprecipitated (IP) with anti-caspase-9 antibody, followed by immunoblotting (IB) with antiApaf-1 (top panel) or anti-caspase-9 antibody (bottom panel). Results are representative
of three Western blots. B and C, inhibition of caspase-9 does not lead to complete
inhibition of cephalostatin 1-induced apoptosis. Cells were either left untreated (CO) or
activated with cephalostatin (CPH; 1 ␮M) or etoposide (ETO; 25 ␮g/ml) in the absence or
presence of the caspase-9 inhibitor zLEHD-fmk (50 ␮M, 1-h pretreatment) for 16 h. Then
caspase-3 activity (B) or apoptosis (C) was quantified as indicated in “Materials and
Methods.” Bars, mean ⫾ SE of three independent experiments performed in triplicate.
ⴱⴱⴱ, P ⬍ 0.001 (ANOVA/Dunnett’s test).
ing and inhibiting caspases (10). To clarify whether Smac/DIABLO
released in response to cephalostatin 1 is able to dissociate XIAP from
caspase-9, we immunoprecipitated Smac/DIABLO (Fig. 7A) and
caspase-9 (Fig. 7B), respectively, and detected bound XIAP by Western blot analysis. Fig. 7, A and B, demonstrates that in untreated cells
(CO), XIAP is associated with caspase-9 but not with Smac/DIABLO.
Cephalostatin (1 ␮M) as well as etoposide (25 ␮g/ml) treatment led to
an association of XIAP with Smac/DIABLO and a dissociation of
XIAP from caspase-9.
Cephalostatin 1 Leads to Structural Changes in Mitochondria.
Mitochondrial swelling and subsequent rupture of the outer mitochondrial membrane are considered one mechanism mediating the release
of proapoptotic mitochondrial molecules such as cytochrome c (22).
Because cephalostatin 1 induces the release of Smac/DIABLO, but
not that of cytochrome c and AIF, we examined the ultrastructural
changes of mitochondria in Jurkat cells after stimulation with 1 ␮M
cephalostatin 1 (16 h). As depicted in Fig. 8, cells treated with
cephalostatin 1 show mitochondria that appear smaller with increased
electron density compared with untreated cells, suggesting a “condensation” of the mitochondrial matrix. The cristae appear blurred. In
close vicinity to the mitochondria, a great number of vesicles, presumably ER-derived, were observed.
DISCUSSION
This is the first report showing that an experimental chemotherapeutic drug triggers a death receptor-independent apoptosis in leukemia Jurkat T cells that uses mitochondria as signaling integrator and
Smac/DIABLO as a main mitochondrial signaling molecule. The
following observations support this view: (a) cephalostatin 1-induced
apoptosis occurred independently of the CD95 death receptor and
caspase-8; (b) cephalostatin 1-triggered DNA fragmentation was significantly reduced after overexpression of the Bcl-xL protein; (c)
despite this fact, cephalostatin 1 did not promote the release of
cytochrome c or AIF (however, it led to an early and intense release
of functionally active Smac/DIABLO that occurred before and independent of caspase activation); and (d) overexpression of Bcl-xL
delayed both Smac/DIABLO release and onset of apoptosis. Although
caspase-9 was activated and released from XIAP in response to
cephalostatin, no apoptosome formation could be detected. Moreover,
caspase-9 did not seem to be the essential initiator caspase in cephalostatin-mediated apoptosis. This unique mitochondria-involving signaling pathway was morphologically characterized by the appearance
of mitochondria with an increased matrix density.
Chemotherapeutic drug-induced cellular stress or damage is sensed
by different cellular organelles, such as mitochondria, the cytoskeleton, the nucleus, the ER, or the plasma membrane. Most signals are
finally integrated by mitochondria that translate these diverse signals
into a common execution pathway by the release of cytochrome c, the
formation of the apoptosome, and, finally, the activation of caspases
(5). The cellular signal generated by cephalostatin 1 seems to use
mitochondria but does not culminate in the common execution pathway triggered by cytochrome c. The cues involved in cephalostatin
1-triggered signaling upstream of mitochondria do not involve the
CD95 death receptor or caspase-8, as shown by Jurkat T cells deficient in these molecules. The requirement of other active caspases
upstream of mitochondria is also unlikely because mitochondrial
release of Smac/DIABLO was not affected by the pan-caspase inhibitor zVAD-fmk. Although the signaling leading to mitochondrial
Smac/DIABLO release in response to cephalostatin 1 has not yet been
identified, it does not include caspase activation. This observation is
in contrast to a recently proposed model in which the release of
Smac/DIABLO from mitochondria is a secondary event following
cytochrome c release and caspase activation (23). These differing
results, however, may be attributable to the different apoptotic stimuli
used.
During stress-induced apoptosis, Smac/DIABLO is released along
with cytochrome c from mitochondria into the cytosol. Although
released cytochrome c contributes to the formation of the apoptosome
and thereby to the initiation of the caspase-9-dependent caspase
cascade, Smac/DIABLO promotes caspase activity by binding to the
XIAP, and probably other IAPs, in a manner that displaces caspases
from their inhibitor XIAP (24, 25). The selective release of Smac/
Fig. 7. In response to the cephalostatin 1 released, Smac/DIABLO sequesters XIAP
bound previously to caspase-9. Cells were left untreated (CO) or stimulated with cephalostatin 1 (CPH; 1 ␮M) or etoposide (ETO; 25 ␮g/ml) for 4 h. A, lysates were immunoprecipitated (IP) with anti-Smac antibody, followed by immunoblotting (IB) with
anti-XIAP (top panel) or anti-Smac antibody (bottom panel). B, lysates were immunoprecipitated (IP) with anti-caspase-9 antibody and analyzed by IB with anti-XIAP antibody (top panel) or anti-caspase-9 antibody (bottom panel). All experiments were performed three times with analogous results. Representative Western blots are shown.
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CEPHALOSTATIN 1-INDUCED APOPTOSIS
Fig. 8. Electron micrographs of untreated Jurkat cells (A)
or Jurkat cells treated with cephalostatin 1 (1 ␮M) for 16 h
(B). The mitochondria (arrows) of the control cells have little
contrast relative to the cytoplasm. Incubation with cephalostatin 1 has two main effects: (a) the mitochondrial matrix
condenses, and the cristae swell, leading to higher contrast of
mitochondria against cytoplasm (Fig. 8B, inset); and (b)
numerous vesicles (asterisks), presumably ER-derived, accumulate in the cytoplasm.
DIABLO by cephalostatin 1 without cytochrome c and subsequent
apoptosome formation are unusual events and raise the following
question: how is caspase-9 activated in the absence of an apoptosome?
Recent data suggest that under certain conditions, Smac/DIABLO
may be sufficient to initiate a caspase-9-dependent caspase cascade in
the absence of cytochrome c and apoptosome formation (26, 27). Our
studies, however, promote the idea that in the absence of an apoptosome, the common mitochondrial execution pathway mediated by the
initiator caspase-9 may not be essential for triggering apoptosis.
A Smac-dependent and apoptosome-independent pathway was recently proposed for receptor-mediated apoptosis in “type II” cells,
including Jurkat T cells. In type II cells, death receptor-mediated
activation of caspase-8 was shown to lead to an incomplete processing
of caspase-3. This inactive form is bound and thus prevented from
autocatalytic processing by XIAP. Smac/DIABLO release was necessary and sufficient to promote full caspase-3 activation (19, 28). In
this setting, Smac/DIABLO clearly functions as a promotor of caspase
activation and not as an inducer. Full caspase-3 activation required
both signaling via caspase-8 and Smac/DIABLO. This gives rise to
the speculation that cephalostatin 1-induced apoptosis that is obviously routed via mitochondria may use other pathways that act in
concert with Smac/DIABLO to fully activate a caspase cascade that
finally leads to apoptosis. It is conceivable that other organelles, such
as the ER, are involved in cellular stress sensing (29, 30). In our
model, overexpression of Bcl-xL blocked both Smac/DIABLO release
and apoptosis, indicating that Smac/DIABLO is a necessary molecule
in cephalostatin 1-induced apoptosis; however, other molecules (located in mitochondria as well as in other organelles) with activities
that are blocked by Bcl-xL may contribute to the activation of caspases
and subsequent apoptosis.
The unique mode of mitochondrial “activation,” i.e., the selective
release of Smac/DIABLO in response to cephalostatin 1, prompted us
to examine putative changes in the mitochondrial morphology. Surprisingly, we found shrunken mitochondria with a higher matrix
density after cephalostatin 1 treatment. This is in contrast to earlier
observations made during “classical” mitochondrial apoptosis (e.g.,
such as that induced by staurosporine in Jurkat T cells). Here, apoptosis was shown to be accompanied by mitochondrial swelling (31).
The observation of mitochondrial swelling during apoptosis is included in one hypothesis explaining how Bcl-2 family members
mediate the release of proapoptotic mitochondrial proteins, such as
cytochrome c: proapoptotic Bcl-2 family members alter mitochondrial
homeostasis, resulting in organelle swelling, physical rupture of the
outer membrane, and subsequent release of intermembrane proteins
into the cytosol (22). This hypothesis obviously does not hold true for
the release of Smac/DIABLO in response to cephalostatin 1 in Jurkat
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CEPHALOSTATIN 1-INDUCED APOPTOSIS
T cells. However, the obvious difference in morphology of mitochondria after treatment with cephalostatin 1 emphasizes a role for mitochondria and that this role differs from that in classical mitochondrial
apoptosis induced by chemotherapeutic drugs and compounds such as
staurosporine.
In summary, we have presented evidence that the marine natural
product cephalostatin 1 triggers apoptosis in leukemia Jurkat T cells
by a novel pathway that promotes the release of Smac/DIABLO but
not cytocrome c or AIF from mitochondria. Although, at present, it
cannot be excluded that one or more other factors are involved that
cooperate with Smac/DIABLO, Smac/DIABLO seems to be required
for cephalostatin 1-induced apoptosis. Of course, strategies to silence
Smac/DIABLO have to be established to finally answer this question.
This unique mechanism is accompanied morphologically by mitochondrial “condensation” instead of swelling.
ACKNOWLEDGMENTS
We thank Drs. Peter H. Krammer and Henning Walczak (German Cancer
Research Center, Heidelberg, Germany) and Dr. Schulze-Osthoff (University
of Düsseldorf, Düsseldorf, Germany) for supplying the used Jurkat T-cell
clones. We also thank S. Dobler and C. Niemann for excellent technical
assistance.
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Research.
Cephalostatin 1 Selectively Triggers the Release of
Smac/DIABLO and Subsequent Apoptosis That Is
Characterized by an Increased Density of the Mitochondrial
Matrix
Verena M. Dirsch, Irina M. Müller, Sören T. Eichhorst, et al.
Cancer Res 2003;63:8869-8876.
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