a-Fetoprotein positively regulates cytochrome c -mediated caspase activation and apoptosome complex formation Lidia Semenkova1,*, Elena Dudich1,*, Igor Dudich1, Natalie Tokhtamisheva1, E
Trang 1a-Fetoprotein positively regulates cytochrome c -mediated caspase activation and apoptosome complex formation
Lidia Semenkova1,*, Elena Dudich1,*, Igor Dudich1, Natalie Tokhtamisheva1, Edward Tatulov2,
Yury Okruzhnov3, Jesus Garcia-Foncillas3, Juan-Antonio Palop-Cubillo4and Timo Korpela5
1
Institute of Immunological Engineering, Moscow, Russia;2Anticancer Drug Research Center, Moscow, Russia; Departments of
3
Oncology and4Organic Chemistry and Pharmacology, University of Navarra, Pamplona, Spain;5Joint Finnish-Russian
Biotechnology Laboratory, Turku University, Finland
Previous results have shown that the oncoembryonic marker
a-fetoprotein (AFP) is able to induce apoptosis in tumor
cells through activation of caspase 3, bypassing
Fas-dependent and tumor necrosis factor receptor-Fas-dependent
signaling In this study we further investigate the molecular
interactions involvedin the AFP-mediatedsignaling of
apoptosis We show that AFP treatment of tumor cells is
accompaniedby cytosolic translocation of mitochondrial
cytochrome c In a cell-free system, AFP mediates
process-ing andactivation of caspases 3 and9 by synergistic
enhancement of the low-dose cytochrome c-mediated
sig-nals AFP was unable to regulate activity of caspase 3 in cell
extracts depleted of cytochrome c or caspase 9 Using
high-resolution chromatography, we show that AFP
posit-ively regulates cytochrome c/dATP-mediated apoptosome
complex formation, enhances recruitment of caspases and Apaf-1 into the complex, andstimulates release of the active caspases 3 and9 from the apoptosome By using a direct protein–protein interaction assay, we show that pure human AFP almost completely disrupts the association between processedcaspases 3 and9 andthe cellular inhibitor of apoptosis protein (cIAP-2), demonstrating its release from the complex Our data suggest that AFP may regulate cell death by displacing cIAP-2 from the apoptosome, resulting
in promotion of caspase 3 activation andits release from the complex
Keywords: apoptosis; apoptosome; cytochrome c; IAP-2; a-fetoprotein
Apoptotic cell death is characterized by biochemical and
morphological changes, which are largely causedby caspase
activity A class of cysteine proteases, known as caspases,
which are constitutively expressedin cells as inactive
proenzymes, require proteolytic cleavage to be activated
In general, either receptor-induced or
mitochondrion-induced death signals stimulate activation of specific
ad apter proteins FADD/MORT1 or Apaf-1 by formation
of the high-molecular-mass death-inducing complex or
apoptosome The adapter proteins recruit initiator caspases
8 and9 to activate them by autoprocessing Once activated,
initiator caspases are ready to induce processing of
down-stream effector caspases 3 and7 [1] The mitochondrial
apoptosis pathway is mediated by cytochrome c (cyt-c)
release with the subsequent formation of the Apaf-1/cyt-c/
dATP/procaspase 9 apoptosome complex, leading to
acti-vation of caspase 9 anddownstream effector caspases [2]
Chromatographic analysis of the apoptosome assembly indicated that, in native cell lysates, Apaf-1 oligomerizes into multimeric complexes of molecular mass 1.4 MDa and 700 kDa, which in addition to processed caspase 9, contain fully processedcaspase 3 and7 [3] Caspases are inhibitedby a number of cellular inhibitor of apoptosis proteins (cIAPs), which binddirectly to procaspases 9 and3
to prevent their cyt-c-mediated processing and activation [4,5] During apoptosis, a mitochondrial protein named Smac/DIABLO [6] that directly binds to IAPs to remove them from the apoptosome complex [4,7], cancels the mediated caspase inhibition Recently, another IAP-inhibitory protein Omi/HtrA2 was characterized, which operates by abrogation of the IAP–caspase interaction [8] AFP is the major serum protein of embryonic plasma that is involvedin regulation of gene expression, differen-tiation, proliferation andapoptosis in developing cells [9–12] Although, the biological role of this protein is not yet fully understood, it has been well characterized as a physiological carrier/transport protein for various ligands, including fatty acids, drugs, hormones, heavy metals, delivering them to developing and malignant cells [9,12] The specific expression andinternalization of AFP is restrictedto developing cells, such as embryonic cells, activatedimmune cells andtumor cells, which suggests its important regulatory role in cell growth anddifferentiation [9,10,12] Various researchers have documented the exist-ence of specific receptor-dependent mechanisms responsible for the active endocytosis of AFP by malignant cells [13,14] Microscopic data have demonstrated that fluoresceinated
Correspondence toE Dudich, Institute of Immunological Engineering,
142380, Lyubuchany, Moscow Region, Chekhov District, Russia.
Tel./Fax: + 7 095 996 15 55, E-mail: dudich@ineos.ac.ru
Abbreviations: AFP, a-fetoprotein; cyt-c, cytochrome c; cIAP, cellular
inhibitor of apoptosis protein; Ac-DEVD-AMC,
Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; LEHD-AFC,
Leu-Glu-His-Asp-aminotrifluoromethylcoumarin; IETD-AMC,
Ile-Glu-Thr-Asp-7-amino-4-methylcoumarin; CHO, aldehyde.
*Note: These authors contributedequally to this work.
(Received11 February 2003, revised28 August 2003,
accepted16 September 2003)
Trang 2AFP is specifically boundto the cell surface at 4C and
internalizedinto the cytoplasm at 37C [15,16] It has been
shown that AFP is internalizedvia coatedpits andvesicles
before being delivered to endosomes [15,16] Much evidence
of cell growth regulatory activity, including tumor
suppres-sion, has been reportedfor various species of the full-length
AFP molecule [17–22], its proteolytic fragments [23],
recombinant domains [24] and synthetic peptides [25–27]
It has been demonstrated that AFP realizes its
tumor-suppressive activity by triggering apoptosis, characterized
by typical morphological changes, growth arrest,
cytotoxi-city, andDNA fragmentation [20–22] It was shown that
AFP induces apoptosis in malignant cells through
activa-tion of caspase 3, bypassing Fas/FasL andtumor necrosis
factor (TNF)/TNFR-dependent pathways and does not
require upstream activation of receptor-dependent initiatory
caspase 8 andcaspase 1 [21] Although these studies have
shown that a caspase cascade is initiated during
AFP-induced apoptosis, the mechanisms by which AFP triggers
caspase activation are unknown Our previous experimental
data show that AFP does not require de novo protein
synthesis andRNA expression to trigger apoptosis, as it was
not blockedby actinomycin D or cycloheximide [20]
In this study, we aimed to determine how AFP activates
the caspase cascade To understand the molecular
mecha-nisms of AFP-mediated apoptosis signaling, we established
a cell-free system, similar to that usedfor studies of
cyt-c-induced apoptosis [28,29] We show here that AFP
syner-gistically enhances caspase activation andprocessing in the
presence of a low suboptimal dose of cyt-c and requires the
presence of all members of the apoptosome complex to
initiate this process We examine the mechanisms by which
AFP regulates apoptosis anddemonstrate that the
pro-apoptotic effect of AFP is mediated through its interaction
with apoptosome-forming proteins Chromatographic
ana-lysis of the apoptosome assembly demonstrated that AFP
stimulates formation of the Apaf-1–apoptosome complex,
enhances recruitment andactivation of procaspase 3 in the
complex, andstimulates release of active caspase 3 and9
from the apoptosome Our data suggest that AFP may
regulate cell death by displacing cIAP-2 from the
apopto-some complex, thereby promoting caspase 3 release from
the complex
Materials and methods
AFP purification
Human AFP was isolatedfrom the cordserum using
ion-exchange, affinity andgel-filtration chromatography as
described previously [23] AFP purity was established using
PAGE andimmunoblotting with monospecific antibodies
against human AFP andadult serum proteins andwas
shown to be no less than 99.8%
Cells
HepG2 cells originatedfrom the American Type Culture
Collection were culturedin Dulbecco modifiedEagle’s
medium (ICN Biomedicals) with L-glutamine and10%
heat-inactivatedfetal bovine serum, 100 IU penicillinÆmL)1,
0.1 mg streptomycinÆmL)1 in a humidified 5% (v/v)
atmosphere of CO2 at 37C For a passage, cells were incubatedin 0.25% (v/v) trypsin solution, then washedand platedout
Cytotoxicity assay HepG2 cells were incubatedwith 5–7 lMAFP for deter-minedtime intervals of 2–14 h, andthen assessedfor their viability by the trypan blue exclusion assay as described previously [22] Cells cultivated without additions were taken as a control The experimental data were expressed as the percentage of dead cells relative to the total amount of cells
Preparation of cell-free extracts Cell-free S-100 extracts were generatedfrom human hepatocarcinoma HepG2 as described [29,30] Cells (4· 108) were collectedandwashed(three times) in
50 mL NaCl/Piandonce in 5 mL hypotonic cell extraction buffer (containing 20 mM Hepes, pH 7.2, 10 mM KCl,
2 mM MgCl2, 1 mM dithiothreitol, 5 mM EGTA, 25 lgÆmL)1leupeptin, 5 lgÆmL)1pepstatin, 40 mM b-glycero-phosphate, 1 mMphenylmethanesulfonyl fluoride) The cell pellet was then resuspended in an equal volume of cell extraction buffer, allowedto swell for 20 min on ice, and then disrupted by 30–50 strokes of a Dounce homogenizer The homogenate was centrifugedat 3000 g for 10 min at
4C to remove whole cells andnuclei The supernatant was centrifugedat 15 000 g for 20 min at 4C and then, to obtain the cytosolic S-100 extract, the supernatant was re-centrifugedat 100 000 g for 1 h at 4C Extracts were assessedfor protein content by the Bradfordassay and storedin aliquots at )70 C Cyt c-free cytosolic extracts were preparedin more mildconditions by the slightly modified procedure described in [30]
In vitro caspase activation For in vitro caspase activation, 40 lg of the S-100 extract (complete or after immunodepletion) was incubated for the indicated times with bovine heart cyt-c (Sigma-Aldrich,
St Louis, MO, USA) and/or pure human AFP (5 lM) in the presence or absence of 1 mMdATP (Sigma) in 15 lL of a reaction buffer (10 mMHepes, pH 7.2, 25 mMNaCl, 2 mM MgCl2, 5 mMdithiothreitol, 5 mMEDTA, 0.1 mM phenyl-methanesulfonyl fluoride) at 30C To control specificity
of AFP effects, the equivalent amount of human serum albumin (Sigma) was added instead of AFP The activity and proteolytic processing of caspases 3 and9 were then detected
by fluorimetric assay andimmunoblotting with the corres-ponding antibodies supplied by Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA): polyclonal goat anti-(caspase 3) p20 (N19); (caspase 3) p11 (K19); rabbit anti-(caspase 9) p10 (H-83); rabbit anti-anti-(caspase 9) p35 (H-170) Fluorimetric assay of caspase activity
Caspase activities were determined by incubation of the extract aliquots (5 lL) for various times at 30C with one
of the fluorogenic substrates [40 lM Ac-DEVD-AMC (ICN Biomedicals Inc), 50 l LEHD-AFC (Chemicon
Trang 3International, Temecula, CA, USA) or 50 lMIETD-AMC
(Alexis Biochemicals, San Diego, USA] in 16 lL substrate
buffer (25 mMHepes, pH 7.2, 100 mMNaCl, 1 mMEDTA,
0.1% Chaps, 10 mMdithiothreitol, 10% sucrose) Reactions
were terminatedby dilution with 2.0 mL ice-cold0.2 mM
sodium phosphate buffer, pH 7.5, and fluorescence was
measuredusing a Perkin–Elmer MPF-44A fluorimeter
(kexc¼ 365 nm and kem¼ 440 nm for the AMC
fluores-cence or kexc¼ 400 nm and kem¼ 505 nm for the AFC
fluorescence) For each sample, caspase activity was
expressedin relative units, pmolÆmin)1Æmg)1, showing the
amount of cleavedsubstrate in pmol normalizedfor time of
reaction with substrates andcytosolic protein
concentra-tion, or in relative fluorescent units (FU) per fraction
Immunoprecipitation and immunoblotting analysis
S-100 cytosolic extracts obtainedfrom HepG2 cells were
immunodepleted from endogenous cyt-c, procaspase 9 or
procaspase 3 by immunoprecipitation with the
corres-ponding antibodies as described [31] Briefly, 50 lL of the
S-100 cell extract (4–5 mgÆmL)1; reaction buffer with
addition of 0.1% Chaps) was incubated for 2 h at 4C
with 5 lg of the corresponding antibodies: anti-cyt-c
6H2.B4 (PharMingen, San Diego, CA, USA),
anti-(caspase 9) clones C-18 andH-83 or anti-anti-(caspase 3)
(N-19) The control cell extracts were incubatedwith the
equivalent amounts of the control antibodies of the same
type Immune complexes were precipitated by addition of
antibody/extract mixture on to drained protein
G-Seph-arose or protein A/agG-Seph-arose beads (Amersham Pharmacia
Biotech) for 2 h at 4C Coatedbeads were then
removedby centrifugation, andthe resulting
immuno-depleted lysates after adjustment for protein concentration
were usedimmediately for caspase activation experiments
The extent of depletion was controlled by
immunoblot-ting with the corresponding antibodies Immunoblotimmunoblot-ting
with b-actin antibodies (ICN Biomedicals Inc) was
performedas a loading control
For immunoblotting analysis, protein samples (50 lg per
lane) were subjectedto standardSDS/PAGE in a 12% or
15% polyacrylamide gel andtransferredon to 0.45-lM
poly(vinylidene difluoride) membranes by semidry
electro-blotting, followedby probing for various proteins using the
corresponding antibodies: rabbit anti-(Apaf-1), H-324
(Santa Cruz); affinity-purifiedrabbit anti-(human cIAP-2),
HIAP-1 (R & D Systems, Wiesbaden, Germany); rabbit
polyclonal anti-(caspase 8) p20, H-134 (Santa Cruz) or the
corresponding polyclonal antibody goat anti-(caspase 3) or
anti-(caspase 9) Bound antibodies were detected using
appropriate horseradish peroxidase-conjugated anti-rabbit
or anti-goat secondary IgGs (Santa Cruz) and developed by
enhancedchemiluminescence staining using ECL reagents
(Amersham Pharmacia Biotech) Gel calibration was
per-formedwith the Low Molecular Weight Calibration Kit for
SDS Electrophoresis (Amersham Pharmacia Biotech)
Dot-blot analysis was performedas usual Briefly, 1-lL
aliquots taken from the chromatographic fractions were
appliedto the nitrocellulose membranes, then blockedby
defattedmilk The membranes were then probedwith rabbit
polyclonal affinity-purifiedanti-(human AFP) IgG Bound
antibodies were detected using appropriate
peroxidase-coupled secondary antibodies and developed as described above
Assay of cyt-c release Cyt-c translocation from mitochondria to the cytoplasm was assessedby direct immunochemical measurement of the cyt-c in the cytosolic andmitochondrial fractions obtained from HepG2 cells treatedwith AFP for various time intervals Briefly, cells (0.5· 106cells per well) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum were platedon the flat-bottomed24-well plates (Nunc) and incubatedfor 24 h Then 5 lMAFP was added to each well After various lengths of treatment (2–17 h), cells were scraped, washed in NaCl/Pi, andresuspendedin 200 lL digitonin lysis buffer (0.025% digitonin in 250 mMsucrose,
20 mM Hepes, pH 7.4, 5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 10 mM Tris/HCl, pH 7.4,
10 lgÆmL)1 leupeptin, 10 lgÆmL)1 aprotinin, and1 mM phenylmethanesulfonyl fluoride) [32] After 10 min, cell lysates were centrifugedfor 2 min at 14 000 g at 4C to obtain the supernatant (cytosolic fraction) andthe pellet (mitochondrial fraction) Mitochondrial pellet was solubi-lizedby a 30-min incubation with 100 lL lysing buffer (150 mMNaCl, 1% Nonidet P40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris/HCl, pH 7.5, cocktail of protease inhi-bitors) Thereafter, cellular debris was removed by a 10-min centrifugation at 14 000 g at 4C The supernatant com-prising the membrane fraction was retained Equal amounts
of cytosolic extracts andsolubilizedmitochondrial pellets (50 lg protein) were fractionatedby SDS/PAGE using 15% polyacrylamide andthen analysedby Western blot using the cyt-c antibody 7H8.2C12, cyt-c oxidase subunit II antibody (Molecular Probes), and b-actin antibody and ECL as described above
Direct protein–protein interaction assay
To determine possible interactions between AFP and caspase 3, caspase 9 andcIAP-2, we useda direct copre-cipitation assay with purifiedproteins Before the experi-ments, 25 lL Ni/Sepharose beads (Qiagen, Valencia, CA, USA) were incubatedfor 1 h at 20C in a solution of assay buffer (50 mMTris/HCl, 100 mMKCl, 10% sucrose, 0.1% Chaps, 0.5 mM dithiothreitol, pH 7.4), containing 1% ovalbumin, 12 lg His-taggedhuman recombinant caspase 9 and3 lg active His-taggedrat recombinant caspase 3 (Alexis Biochemicals) After being washed, one half of the beads was added to the cytosolic extract of HepG2 cells (500 lg total protein) together with 20 lg AFP and incubated for 2 h at 4C The control beads were incubatedwith the same amount of HepG2 cytosolic extract without AFP addition The protein–bead complexes were then washed(four times), isolatedby centrifugation, boiled
in 15 lL sample buffer, andanalyzedby SDS/PAGE/ Western blotting with anti-cIAP2 (HIAP-1) IgG
Chromatographic analysis of the apoptosome assembly
To study effects of AFP on recruitment, processing and release of various caspases from apoptosome andmicro-apoptosome complexes, we usedthe previously described
Trang 4gel filtration technique [3] Briefly, S-100 extracts were
preparedfrom HepG2 cells (6 mgÆmL)1) andactivatedby a
1-h incubation at 30C with 1.0 mMdATP/1.5 mMMgCl2/
1.0 lMcyt-c with or without 5.0 lMAFP Before addition
to the S-100 extracts, AFP samples were dialyzed against the
elution buffer Activatedlysate proteins ( 1 mg) were
applied(0.2 mLÆmin)1; 4C) to a 10/30 Superose-6 HR
column connectedto an FPLC system (Amersham
Phar-macia Biotech) The column was elutedwith elution buffer
(20 mM Hepes/KOH, 10 mM KCL, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol, 1.5 mM MgCl2, 0.01 mM
phenylmethanesulfonyl fluoride, pH 7.2); 1-mL fractions
were collected Aliquots of the fractions were taken for
measurement of caspase activity using the corresponding
fluorogenic substrates: DEVD-AMC for caspase 3
andAc-IETD-AMC for caspases 9 and8 [33] as describedabove
Fractions were then concentrated20-foldwith 2 mL
centrifugal concentrators (Centricon YM-10; Amicon) and
analyzedby PAGE andimmunoblotting for changes in
distribution of AFP, Apaf-1, cIAP-2, caspases 3, 9 and 8
Column calibration was performedwith Gel Filtration
LMW andHMW calibration kits (Amersham Pharmacia
Biotech)
Results
AFP induces release of mitochondrial cyt-c in HepG2 cells
Our previous publications were devoted to the study of
AFP-inducedapoptosis in whole cells andsuggestedthat
this mechanism is independent of membrane receptor
signaling [20–23] We investigate here the intracellular
molecular pathways of the AFP-mediated triggering of
apoptosis To analyse the involvement of cyt-c release in
AFP-mediatedapoptosis, cytosolic andmitochondrial
fractions were obtainedfrom AFP-treatedHepG2 cells
andanalysedby Western blot for the presence of cyt-c As
shown in Fig 1, AFP induced the appearance of cyt-c in the
cytosolic fraction of treatedHepG2 cells andits
disappear-ance from the mitochondrial fraction of treated cells,
indicating that AFP induced mitochondrial cyt-c release
These data do not show, however, whether AFP induces
cytosolic cyt-c release directly or by indirect mechanisms by
activation of unknown factors
AFP synergistically enhances low-dose cyt-c-mediated
caspase activation in cell-free cytosolic extracts
The mitochondrial apoptotic pathway could be activated by
addition of dATP to cell extracts to initiate the Apaf-1/
procaspase 9/cyt-c apoptosome cascade [28] To determine
whether AFP is involvedin this process, we establisheda
typical cell-free system using HepG2 cells andmeasured
caspase activation in this system with or without addition
of AFP Two types of cell lysate were usedfor these
experiments: a typical S-100 cytosolic extract anda
cyt-c-free cytosolic extract, preparedby a mildprocedure as
described previously [30] Addition of AFP to the S-100
cytosolic extract triggered dATP-dependent induction of
caspase 3-specific DEVDase activity, which progressively
increasedfor at least 2 h (Fig 2A) As a control, the
equivalent amount of human serum albumin was added to
the same cell-free system No effect was observedat the level
of DEVDase activity A low level of DEVDase activity was also induced by dATP alone, evidently due to the presence
of a small amount of endogenous cyt-c in the preparations
In the absence of dATP, AFP did not induce any caspase 3-specific DEVDase activity at all
To determine whether AFP can directly induce caspase activation in cell-free cytosolic extract or requires the presence of the basal level of cyt-c, we examinedDEVDase cleavage activity after addition of exogenous cyt-c and AFP
to the silent cytosolic extracts with undetectable endo-genous cyt-c Figure 2B shows that no DEVDase activity was detected in this type of cytosolic lysate stimulated with dATP/AFP or with dATP and low suboptimal dose of cyt-c even 1.5 h after treatment A significant time-dependent increase in DEVDase activity was observedin the same reaction system only after addition of all three compounds: AFP, d ATP and cyt-c (Fig 2B) The low DEVDase activity
in this experimental system comparedwith that describedin Fig 2A is explainedby the negligible amount of cyt-c in the cytosol These data demonstrate the ability of AFP to amplify caspase-activating signals induced by low subopti-mal doses of cyt-c
We then examinedthe effect of AFP on the DEVDase activity mediated by different doses of cyt-c in S-100 extracts Figure 2C shows that, similarly to the above data (Fig 2A), AFP synergistically enhances DEVDase activity induced by low suboptimal doses of cyt-c A further increase
in cyt-c concentration in the cell extract resultedin the
saturation effect, when the maximal stimulation of caspase 3-specific DEVDase activity was reached, which AFP cannot further increase (Fig 2C)
Fig 1 Effect of AFP on cell viability and cyt-c release in HepG2 cells HepG2 cells were treatedwith 5 l M AFP for various time intervals, andthen cytosolic andmitochondrial extracts were preparedat the indicated times Equal amounts of cytosolic and mitochondrial extracts (50 lg) were immunoblottedwith anti-(cyt-c) to assess cyt-c release b-Actin andcytochrome oxidase subunit II (Cyt ox.) were also analysedin cytosolic andmitochondrial extracts as controls for protein loading Cell viability of AFP-treated HepG2 cells was assessed by the trypan blue exclusion assay as describedin Materials andmethods.
Trang 5AFP synergistically enhances cyt-c-mediated processing
and activation of procaspases 9 and 3 in cell-free
cytosolic extracts
To determine whether AFP could induce increased caspase
activation in a cell-free system, we examinedS-100 extracts
for cleavage of procaspases 3 and9 andcorresponding
fluorogenic caspase substrates after addition of AFP/cyt-c/ dATP Both procaspase 9 and procaspase 3 were processed
to their active forms, giving the corresponding fragments p35/37 andp10 for caspase 9 andp17 andp12 for caspase 3 However, when AFP was combinedwith cyt-c/ dATP, more complete cleavage of the procaspases was observed (Fig 3B,C) In addition, there was a dramatic increase in caspase 3-like DEVDase activity anda notable increase in caspase 9-like LEHDase activity on combined treatment with AFP/cyt-c/dATP in comparison with cyt-c/ dATP (Fig 3A) These data show that AFP positively regulates both processing andactivation of procaspases 9 and3 in cell-free cytosolic extracts by amplification of the low-dose cyt-c-mediated effects
AFP induces caspase activation only in the presence
of the all components of the apoptosome complex The above experiments demonstrated functional interfer-ence of AFP with the cyt-c-mediated process of caspase activation We studied further the functional significance of
Fig 2 AFP enhances cyt-c-mediated DEVDase activity in cell-free
cytosolic extracts (A) AFP induces caspase 3 activation in cell-free
S-100 cytosolic extracts in the presence of dATP Effect of endogenous
cyt-c Aliquots of HepG2-derived cytosolic extract (25 lg protein)
were treatedfor various times with AFP (5 l M ) or as a control with the
same dose of human serum albumin in the presence of dATP (1 m M )
andthen assayedfor DEVDase activity (B) Synergistic increase in
DEVDase activity mediated by AFP in cyt-c-free cytosolic extracts on
addition of exogenous cyt-c Aliquots of the cyt-c-free HepG2-derived
cytosolic extracts (25 lg protein) were treatedfor various times with
AFP (5 l M ), cyt-c (0.2 l M ) or a combination of the same doses of the
two compounds in the presence of dATP (1 m M ) andthen assayedfor
DEVDase activity (C) AFP differently affects caspase 3 activation in
cell-free cytosolic extracts induced by various doses of cyt-c Aliquots
of S-100 cytosolic extract (25 lg protein) were treatedfor 30 min with
AFP (5 l M ) andvarious doses of cyt-c in the presence of dATP (1 m M )
andthen assayedfor DEVDase activity The mean ± SD from four
determinations is shown.
Fig 3 AFP positively regulates cyt-c-mediated DEVDase and LEH-Dase activity and processing of procaspase 9 and 3 in a cell-free system Aliquots of HepG2-derived S-100 cytosolic extract with addition of
1 m M dATP were treated in the presence (+) or absence (–) of cyt-c (0.2 l M ) and/or AFP (5 l M ) (A) Proteolytic activities of caspase 9 and
3 in experimental lysates were assayedby monitoring the cleavage of the corresponding fluorogenic substrates LEHD-AFC and Ac-DEVD-AMC The mean ± SD from four determinations is shown Processing of caspases was detected by immunoblotting with the cor-responding antibodies that recognize the precursors and subunits of active caspase 9 (B) and3 (C).
Trang 6AFP in regulation of activity of the apoptosome complex.
Cellular extracts were sequentially depleted of the main
active molecular compounds involved in the formation of
the apoptosome complex: endogenous cyt-c, procaspase 3,
or procaspase 9 Caspase activation was then induced by the
addition of cyt-c/dATP with or without AFP AFP was
unable to induce caspase 3 activation in the absence of cyt-c
and/or dATP in the cyt-c-immunodepleted cytosolic extracts
(Fig 4) However, addition of exogenous cyt-c together with
dATP produced DEVDase activity Simultaneous addition
of all three compounds (AFP, cyt-c and dATP) resulted in
significant enhancement of total DEVDase activity
com-paredwith that inducedwith cyt-c/dATP (Fig 4)
We next determined whether AFP requires the presence
of procaspase 9 to induce caspase 3 activation mediated by
a suboptimal dose of cyt-c HepG2 S-100 extracts were
depleted of procaspase 9 by immunoprecipitation with the
corresponding antibody and then treated with AFP/cyt-c or
cyt-c alone in the presence of dATP Figure 5A,B shows
that removal of caspase 9 from cell extracts ledto the
complete loss of AFP/cyt-c-mediated DEVDase activity,
whereas control extracts andextracts treatedwith anti-RXR
(antibody control) displayed significant enhancement of the total cyt-c-mediated DEVDase activity in response to AFP addition These results are supported by additional data showing that the specific caspase 9 inhibitor Ac-LEHD-CHO significantly suppressed cyt-c/dATP-dependent AFP-mediated DEVDase activity in a cell-free system (Fig 5B) The results show that AFP with or without cyt-c cannot directly induce caspase 3 activation in a cell-free system in the absence of procaspase 9
AFP cannot induce activation of procaspase 9
in the absence of caspase 3
As AFP was unable to activate procaspase 3 in the absence
of procaspase 9, we further studied whether AFP is capable
of activating procaspase 9 independently of caspase 3 HepG2-derived S-100 cytosolic extracts were depleted of procaspase 3 by immunodepletion with the corresponding
Fig 4 Depletion of cyt-c abrogates AFP-mediated caspase activation in
cytosolic extracts (A) Endogenous cyt-c was removed from S-100
cytosolic extract by immunoprecipitation with anti-cyt-c mAb 6H2.B4.
To confirm cyt-c depletion, equal amounts (50 lg) of control untreated
extract, extract treatedwith unspecific mouse IgG (antibody control)
andcyt-c-depletedextract were resolvedby SDS/PAGE
andimmu-noblottedwith anti-(cyt-c) b-Actin was usedas a loading control (B)
Caspase activation in cyt-c-depletedlysate was inducedby treatment
with appropriate doses of AFP (5 l M ) and/or cyt-c (0.2 l M ) in the
presence of dATP (1 m M ) Caspase 3 activity was measuredby
monitoring cleavage of the fluorogenic substrate DEVD-AMC The
mean ± SD from four determinations is shown.
Fig 5 Procaspase 9 is required for AFP-mediated caspase 3 activation (A) S-100 cytosolic extract was immunodepleted of procaspase 9 by immunoprecipitation with anti-(caspase 9) To confirm caspase 9 depletion, equal amounts (50 lg) of control untreatedextract, cyt-c-treatedextract, extract treatedwith anti-RXR (control for possible unspecific antibody-induced effects) and caspase 9-depleted extract were analysedby immunoblotting with anti-(caspase 9) b-Actin was usedas a loading control (B) Caspase 3 activation was inducedin different types of experimental extract: caspase 9-depleted extract, complete extract, complete extract incubatedwith Ac-LEHD-CHO andextract treatedwith anti-RXR Extracts were activatedby addition (+) or in the absence (–) of appropriate doses of AFP (5 l M ) and /or cyt-c (0.2 l M ) in the presence of dATP (1 m M ) Caspase 3 activity was measuredby cleavage of the fluorogenic substrate DEVD-AMC The mean ± SD from four determinations is shown.
Trang 7antibody Depletion was controlled by immunoblotting
(Fig 6A) anddirect measurement of the DEVDase activity
(not shown) Thereafter procaspase 3-depleted S-100
extracts were testedfor LEHDase activity upon treatment
with AFP and/or cyt-c Addition of cyt-c to caspase
3-depleted extracts induced a distinct increase in LEHDase
activity, showing caspase 9 activation (Fig 6B) These data
indicate that cyt-c induced dose-dependent activation of
caspase 9 in a caspase 3-independent manner,
demonstra-ting the hierarchical advantage of caspase 9 in this process
In contrast, treatment of caspase 3-depleted extracts with
AFP did not induce any enhancement of LEHDase activity
comparedwith the effect of cyt-c alone (Fig 6B), showing
that the presence of procaspase 3 is critical for the
realization of AFP-mediated pro-apoptotic activity
AFP positively regulates cyt-c-mediated apoptosome
complex formation in a cell-free system and release
of active caspases from the complex
Our current data demonstrate that AFP requires the
presence of all of the main members of the apoptosome
complex (cyt-c, dATP, caspases 9 and 3) to induce caspase
activation in a cell-free system We reasonedthat AFP may
be involvedin regulating the activity of the apoptosome complex To test this hypothesis, we studied the formation
of the apoptosome complex in cell-free extracts induced
by cyt-c/dATP in the presence or absence of AFP by monitoring the distribution of caspase activity along the chromatography pattern To evaluate caspase 8 and caspase 9 activation, we measuredIETDase cleavage activity Figure 7A shows that caspase 8 is completely absent from the position of the active 700-kDa complex (fractions 8–10) andwas detectedonly in fractions 14–15, corresponding to the free form of the processed enzyme, as described previously [31,33] Thus, in the absence of caspase 8 in the apoptosome complex, IETDase cleavage activity in this region may represent effects induced by active forms of caspase 9 [34] The data obtained from measurement of LEHDase cleavage activity showedsigni-ficantly lower fluorescent intensity andwere difficult to interpret (not shown) Our data demonstrate that AFP did not induce any changes in IETDase activity in the position
of the active 700-kDa complex (fractions 8–10), but DEVDase activity in this region was notably enhanced comparedwith the effect of cyt-c alone (Fig 7A,B) The most significant AFP-mediated increase in DEVDase cleavage activity was observedat 70–60 kDa (fractions 15–17), corresponding to the free active caspase 3 (Fig 7B) Figure 7A shows that integral IETDase activity
at 90 kDa corresponding to free active caspases 9 and 8 (fractions 14–15) was also enhanced after AFP addition (Fig 7A)
The distribution of caspase 9 and caspase 3 precursors andmature forms distinctly correlates with the corres-ponding activity patterns (Fig 7A,B) Caspase 9 was processedunder these conditions andshowedtwo peaks
in the column for both experimental systems with and without addition of AFP The main peak of caspase 9-specific material was locatedin fractions 9–10, whereas the secondpeak was at fractions 13–15 It shouldbe mentionedthat a smaller amount of the processed caspase 9 was also detected in fractions 6–7, correspond-ing to the biologically inactive 1.4-MDa apoptosome complex (not shown), similarly to previously reported data [3,35] Our data confirmed results obtained by these authors [3,35] indicating that in spite of the presence of all of the members of the apoptosome complex (Apaf-1, cyt-c, caspase 9) in the 1.4-MDa apoptosome complex,
it was unable to cleave IETD-like substrates, showing its inability to process effector caspases In the absence of AFP, the precursor of caspase 9 was recoveredmainly in the free form in fractions 14–15, demonstrating that a low suboptimal dose of cyt-c does not recruit all the available procaspase 9 for apoptosome formation A small amount of processedcaspase 9 was also foundin this case in fraction 13 corresponding to a molecular mass of 160–180 kDa, indicating the formation of an intermediate complex (Fig 7A, bottom) After treatment
of S-100 with AFP/cyt-c/dATP, we observed a significant increase in the total amount of the processedcaspase 9
in fractions 14–15, indicating that AFP stimulates both maturation of caspase 9 andits release from the complex
In the S-100 extract, which was stimulatedwith cyt-c/
dATP, both precursor andprocessedforms of caspase 3
Fig 6 AFP cannot induce activation of procaspase 9 in the absence of
caspase 3 (A) Procaspase 3 was immunodepleted from S-100 extracts
by immunoprecipitation with anti-(caspase 3) To confirm
immuno-depletion, 50 lg protein from control complete extract, extract treated
with goat IgG (control for possible unspecific antibody-induced
effects) andcaspase 3-d epletedextract were analyzedby
immuno-blotting with anti-(caspase 3) b-Actin was usedas a loading control.
(B) Caspase 9 activation in caspase 3-depleted extracts was induced by
addition of the appropriate doses of AFP (5 l M ), cyt-c (0.2 l M ), and
dATP (1 m M ) andassessedby cleavage of the fluorogenic substrate
LEHD-AFC The mean ± SD from four determinations is shown.
Trang 8were detected mainly in fractions 13–14 ( 160–
180 kDa), reflecting activity distribution (Fig 7B) These
data indicate that, at low cyt-c, caspase 3, like caspase 9,
tends to form an intermediate 160–180-kDa complex
or migrate together with other protein aggregates in this
region In the extracts stimulatedwith AFP/cyt-c/dATP,
we revealedthe precursor form of caspase 3 in fractions
13–14, whereas processedcaspase 3 was recovered
mainly in fractions 15–16, showing again that AFP
stimulates release of the free active caspase 3 from the
complex
We have also monitoredthe distribution of AFP along the chromatography pattern of the S-100 extracts after addition of AFP/cyt-c/dATP and found this 70-kDa protein
in fractions corresponding to the high-molecular-mass complexes (Fig 7B, bottom) Pure protein migrates at the position corresponding to its monomeric size compared with the molecular mass standard This indicates that AFP may be involvedin formation of the high-molecular-mass multimeric complexes with cytosolic proteins and may modulate protein–protein interactions within the complexes
Fig 7 AFP positively regulates formation of Apaf-1 apoptosome in cell-free extracts and promotes caspase activation and release of caspase 3 and 9 fromthe complex Aliquots (1 mL) of S-100 extracts obtainedfrom nonapoptotic HepG2 cells were left untreated(control) or activatedby a 1-h incubation at 30 C with 1 m M dATP and 0.7 l M cyt-c in the presence or absence of 5.0 l M AFP Subsequently, the extract aliquot (1 mg protein) was fractionatedby high-resolution chromatography on a Superose-6 HR 10/30 column Fractions of 1 mL were collectedandaliquots of 50 lL were assayedfluorimetrically for IETDase (A) andDEVDase (B) activity Caspase activity is given in arbitrary fluorescent units in the fraction per minute Arrowheads at the top of the patterns indicate sizes of calibration protein standards and their elution positions from the Superose-6 column Dot-blot analysis of the AFP distribution in the fractions for cell lysates treated with AFP/dATP/cyt-c is shown under the chromatographic pattern (B) The corresponding fractions were concentrated, and aliquots of 20 lL were also resolvedby SDS/PAGE andimmunoblotting for caspase 9, caspase 8 (A), caspase 3 (B), Apaf-1 (C) and anti-(cIAP-2) (D) The corresponding chromatography fraction numbers are indicated under the patterns The central line marked with an asterisk shows the blot of cell lysate with addition of cyt-c/dATP before chromatography (E) AFP displaces endogenous cIAP-2 from the complex with caspases 3 and9 Recombinant His-taggedactive caspase 9 andcaspase 3 were immobilized
on the Ni/Sepharose beads andincubatedwith HepG2 S-100 extract with or without 5 l M AFP Ni/Sepharose-boundproteins were analyzedby SDS/PAGE/immunoblotting with polyclonal antibodies to cIAP-2.
Trang 9Effect of AFP on the distribution of Apaf-1 and cIAP-2
proteins along the chromatographic pattern
of the apoptosome assembly
To determine possible mechanisms of the AFP-mediated
regulation of the apoptosome complex, we monitoredthe
distribution of Apaf-1 along the chromatographic pattern
of the apoptosome assembly, which was formedwith and
without AFP (Fig 7C) In cell extracts stimulatedwith low
cyt-c, Apaf-1 was recoveredin two main peaks
correspond-ing to fractions 6–8 and13–15, demonstratcorrespond-ing that a low
suboptimal dose of cyt-c does not recruit all the available
Apaf-1 into the functional apoptosome andtends to form
the nonfunctional complex of molecular mass 1.4 MDa
In the presence of AFP, Apaf-1 specificity was significantly
reduced in the biologically inactive 1.4-MDa complex
(fractions 6–7) [3,35], but notably increasedin the region of
the 700-kDa apoptosome (fractions 8–10) These data
indicate that, at low cyt-c, AFP positively modulates
recruitment of Apaf-1 into the active 700-kDa
apopto-some complex
Figure 7D shows that cIAP-2 distribution was not so
clearly affected by AFP addition as observed in the case of
Apaf-1 However, in the absence of AFP, full-length cIAP-2
was present in fractions 10–11, whereas fraction 9 mainly
containedfragmentedIAP-2-specific material (Fig 7D)
After the addition of AFP, the cIAP-2 specificity (including
full-length protein and its fragments) was distinctly reduced
in fractions 9–10 (Fig 7D) The similar fragmentation
pattern for cIAP-1 andcIAP-2 has been
describedprevi-ously [36] It was shown that fragmentedcIAP-1 andcIAP-2
were more effective at protecting cells from apoptosis,
whereas full-length proteins lackedprotective activity
Removal of the RING domain by proteolysis restored the
antiapoptotic activity [36] It was also shown that cIAP-1
was cleaved in vitro by pure caspase 3, producing similar
52-kDa and35-kDa fragments Our data allow us to suggest
that AFP may negatively regulate fragmentation of cIAP-2,
thus modulating its antiapoptotic activity Alternatively,
AFP may stimulate release of active fragmentedcIAP from
the apoptosome From our data we proposed the possible
interaction of AFP with cIAP-2 andits partial removal from
the apoptosome complex To confirm this, we studied the
direct interaction of AFP and cIAP-2 using a direct protein–
protein interaction assay
Interaction between caspase 9, caspase 3, c-IAP-2
and AFP
To study further the interaction between AFP, cIAP-2 and
caspases 9 and3, we precipitatedpure recombinant active
caspases 3 and9 (His-tagged) with nickel resin andthen
incubatedthem with AFP andS-100 extract, as a source of
cIAP-2 A similar reaction mixture was also prepared
without AFP The supernatants andpellets were probed
with antibodies against cIAP-2 As IAPs interact directly
with active caspases 3 and9 [5], we speculatedthat AFP
may physically interact with one of these proteins to
displace cIAP-2 from the complex Figure 7E shows that
cIAP-2 binds processed recombinant caspase 3 and/or 9
Addition of the pure human AFP in the same reaction
system almost completely disrupts the interaction between
processedcaspases andcIAP-2, demonstrating its release from the complex (Fig 7E) Additional experiments with each protein member of the complex will be necessary to clarify the exact molecular interactions involvedin this effect Our data suggest that AFP may positively regulate the activity of the apoptosome by negative modulation of the cIAP-2 content, resulting in promotion of the release of active caspases 3 and9 from the complex
Discussion
There is increasing evidence that AFP may selectively induce activation of programmed cell death in tumor cells [17–23], showing its potential for cancer treatment [10] Various researchers have documented the tumor-selective uptake of AFP by malignant cells [13–16], but the functional significance of this phenomenon has not been clarified The exact molecular mechanisms of AFP-mediated apoptosis also remain unclear The present data explain some details
of the molecular interactions in this effect
In this study we have investigated the ability of AFP to directly activate the death program in a cell-free model of apoptosis Release of cyt-c into the cytoplasm of AFP-treated cells suggests that a mitochondrion-dependent mechanism of apoptosis signaling is involved However, these data do not exclude the possibility that another cyt-c-independent pathway of AFP-mediated signaling of apop-tosis is also involved in the sequential indirect induction of cyt-c release with the onset of its activity We foundhere that AFP promotes low-dose cyt-c/dATP-mediated pro-cessing andactivation of procaspases 9 and3 in a cell-free system These data show that AFP is directly involved in regulating the mechanisms of caspase cascade activation andsuggest that it may be involvedin regulating apopto-some complex formation We have demonstrated further that AFP-mediated signaling of apoptosis requires the presence of all the major members of the apoptosome complex: cyt-c, dATP, caspases 9 and 3 To confirm that AFP is involvedin regulating activity of the apoptosome complex, cell-free cytosolic extracts were activated in vitro
by addition of cyt-c/dATP or AFP/cyt-c/dATP, and, after high-resolution gel filtration, the fractions from the column were analysedby Western blotting Our data clearly show that AFP positively regulates cyt-c/dATP-mediated forma-tion of the active 700-kDa Apaf-1–apoptosome complex andstimulates release of the active caspase 3 from the complex The key was the finding that AFP negatively regulates binding of cIAP-2 to active caspases 9 and 3 It remains to be seen if AFP associates with andinhibits interaction of other cIAPs with caspases, thus promoting caspase activation within the apoptosome complex Our data suggest that AFP interacts with caspase 3, 9 and/or cIAP-2 in a similar manner to DIABLO/Smac or Omi/Htr [7,8], but the exact molecular determinants involved in these interactions remain to be determined
A similar effect of selective triggering of apoptosis in tumor cells was observedfor multimeric forms of human a-lactalbumin, MAL [37,38] It was shown that only oligomerizedforms of this protein are capable of inducing apoptosis Our recent data similarly showed that AFP requires concentration-dependent oligomerization to become apoptotically active [23] The exact mechanism of
Trang 10MAL function has not yet been established, but it was
shown that MAL induces its proapoptotic effects by direct
activation of the caspase cascade independently of the
membrane-receptor signaling [38]
To prevent uncontrolledproliferation of rapidly growing
tissues, such as developing immature immune cells,
embry-onic cells or tumor cells, certain natural control mechanisms
have to exist that select and direct developing cells toward
maturation andprevent their neoplastic transformation
This study describes a naturally occurring protein, the
expression of which is restrictedby developing immature
embryonic cells or cells undergoing malignant
transforma-tion [9–12] Proteins with quite mundane functransforma-tions in
healthy cells often behave very differently during cell suicide
The selective proapoptotic activity of AFP, targeting only
neoplastic [17–23] andactivatedimmune cells [9,10],
indi-cates that it is a natural effector in a fetoembryonic defense
system to prevent malignant transformation of developing
cells Our data allow us to propose that AFP helps cells to
overcome their resistance to apoptosis by significant
ampli-fication of the apoptotic signals induced by other factors,
such as drugs and oxidative stress AFP may help cells,
which are resistant to apoptotic stimuli for any reason, to
overcome their resistance, which is induced, for example, by
overexpression of heat shock proteins, cIAPs or any other
defects of apoptosome-dependent apoptotic pathways
Tumor cells are characterizedby defects in expression of
apoptosis-promoting proteins, such as Apaf-1 andp53, and
simultaneous overexpression of the antiapoptotic proteins
Hsp70, Bcl-2 andBcl-xL, resulting in tumor-specific
sup-pression of apoptosis andenhancement of the malignance
andtherapy resistance of tumors [39–43] The existence of a
high backgroundlevel of antiapoptotic factors in the cytosol
of tumor cells often leads to their resistance to apoptosis
induced by weak stress stimuli It has been demonstrated
that high levels of AFP in maternal serum during pregnancy
were associatedwith a low incidence of breast cancer [44,45]
It was proposedthat AFP may delete immature breast
tissue cells that show the first signs of neoplastic
transfor-mation Our results correlate with these data, indicating that
AFP may function to remove transformedneoplastic cells
by tumor-selective amplification of weak apoptotic signals
Of special interest in cancer research are tumor-specific
factors that regulate apoptosis in tumor cells which function
at a common part of the apoptotic signaling pathway and
may cancel their resistance to apoptosis There are several
hypotheses that may help to explain these findings The
release of cyt-c from the mitochondria into the cytosol has
been shown to be one of the earliest apoptotic events, which
occurs before mitochondrial depolarization, caspase
activa-tion andDNA fragmentaactiva-tion It has been documentedthat,
after apoptosis signaling, cyt-c is releasedfrom the
mito-chondria within 5 min [46] The extramitomito-chondrial cyt-c
has been shown to be a general apoptogen in cells with a
functional caspase system [47] On the other hand, recent
papers have shown that cells can survive with reduced
mitochondrial membrane potential and released cytosolic
cyt-c given appropriate signals to suppress apoptosis [48,49]
It was observedthat the amount of releasedcyt-c in K562
andCEM lines didnot correlate with the extent of apoptosis
in response to UV light, showing reduced caspase 3
activa-tion The effect was explainedby the reducedexpression
of Apaf-1 protein in resistant leukemic cells [48] A high backgroundlevel of cytosolic cyt-c has been shown in vivo in the aging heart, with a significant decrease in the antiapop-totic protein bcl-2 [49] In certain types of cancer cell, alterations in the regulation of apoptosis may contribute to tumor malignancy andresistance to radiotherapy and chemotherapy [50] Sometimes dysfunctional apoptosome activation in tumor cells is observedin the presence of the requiredamount of cytosolic cyt-c, dATP, Apaf-1 andpro-caspase 9, leading to significant enhancement of their resistance to apoptotic stimuli including radiotherapy and chemotherapy [51]
Our data indicate that AFP can be considered as a tumor-specific regulator of cyt-c-mediated apoptotic signals
In vivo, it may operate as a specific regulator of the apoptosome dysfunction induced by the impaired release of apoptogenic factors in the cytosol and/or the increased level
of cytosolic antiapoptotic proteins It may operate to amplify weak apoptotic signals induced by oxidative stress, ionizing radiation or drugs to sensitize tumor cells to chemotherapy It seems to operate as a tumor-specific regulator of apoptosis inhibitory proteins, but it remains to
be seen if it associates with andinhibits cIAPs other than cIAP-2, andto determine the molecular mechanisms of these interactions
Acknowledgements
This work is supportedin part by the International Science & Technology Center, ISTC (grants Nos 401-98 and1878-01) We thank
Dr Alex Sazonov for the invaluable gift of recombinant caspase 3 and caspase 9 andDr Alex Chugunov for excellent assistance with FPLC chromatography.
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