Báo cáo khoa học: Alpha-fetoprotein antagonizes X-linked inhibitor of apoptosis protein anticaspase activity and disrupts XIAP–caspase interaction ppt

Alpha-fetoprotein antagonizes X-linked inhibitor of
apoptosis protein anticaspase activity and disrupts
XIAP–caspase interaction
Elena Dudich
1,2
, Lidia Semenkova
1,2
, Igor Dudich
1,2
, Alexander Denesyuk
3
, Edward Tatulov
2
and Timo Korpela
4
1 Institute of Immunological Engineering, Lyubuchany, Russia
2 JSC BioSistema, Moscow, Russia
3 Department of Biochemistry and Pharmacy, A
˚
bo Akademi University, Turku, Finland
4 Joint Biotechnology Laboratory, Turku University, Finland
Apoptotic dysfunction plays a key role in cancer pro-
gression and leads to chemotherapeutic and radio-
therapeutic resistance [1–3]. Many cancer therapeutic
agents operate by inducing apoptosis and are ineffec-
tive in conditions of impaired apoptosis signaling.
Novel strategies for cancer therapy are aimed at dis-
covering molecular targets involved in the induction
of apoptosis in normal and tumor cells, and at selec-
tively regenerating the apoptosis propensity in cancer
cells.

Apoptosis is induced by two different mechanisms:
the extrinsic or receptor-dependent pathway and the
intrinsic or mitochondria-dependent pathway [4]. Trig-
gering of either pathway results in the initiation of
caspase cascade activation events. Caspases are gener-
ally divided into two groups according to their func-
tional hierarchy and substrate specificity. The initiator
caspase family includes caspases 2, 8, 9, 10 and 12,
and is characterized by the presence of N-terminal
prodomains DED or CARD, which are involved in
Keywords
apoptosis; apoptosome; caspases;
a-fetoprotein; X-linked inhibitor of apoptosis
protein
Correspondence
E. Dudich, Institute of Immunological
Engineering, 142380, Lyubuchany, Moscow
Region, Chekhov District, Russia
Fax ⁄ Tel: +7 095 996 1555
E-mail:
(Received 28 February 2006, revised 3 May
2006, accepted 22 June 2006)
doi:10.1111/j.1742-4658.2006.05391.x
Previous results have shown that the human oncoembryonic protein a-feto-
protein (AFP) induces dose-dependent targeting apoptosis in tumor cells,
accompanied by cytochrome c release and caspase 3 activation. AFP posi-
tively regulates cytochrome c ⁄ dATP-mediated apoptosome complex forma-
tion in a cell-free system, stimulates release of the active caspases 9 and 3
and displaces cIAP-2 from the apoptosome and from its complex with
recombinant caspases 3 and 9 [Semenkova et al. (2003) Eur. J. Biochem.

270, 276–282]. We suggested that AFP might affect the X-linked inhibitor
of apoptosis protein (XIAP)–caspase interaction by blocking binding and
activating the apoptotic machinery via abrogation of inhibitory signaling.
We show here that AFP cancels XIAP-mediated inhibition of endogenous
active caspases in cytosolic lysates of tumor cells, as well as XIAP-induced
blockage of active recombinant caspase 3 in a reconstituted cell-free sys-
tem. A direct protein–protein interaction assay showed that AFP physically
interacts with XIAP molecule, abolishes XIAP–caspase binding and rescues
caspase 3 from inhibition. The data suggest that AFP is directly involved
in targeting positive regulation of the apoptotic pathway dysfunction in
cancer cells inhibiting the apoptosis protein function inhibitor, leading to
triggering of apoptosis machinery.
Abbreviations
Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin; AFP, a-fetoprotein; IAP, inhibitor of apoptosis protein; IBM, IAP-binding
motif; RFU, relative fluorescence units; XIAP, X-linked inhibitor of apoptosis protein.
FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3837
interactions with certain adapter molecules to form
death-inducing signaling complex or apoptosome [3–5].
Effector caspases 3, 6 and 7 exist in the cytosol as
inactive zymogens and are activated via a proteolytic
cascade started by the initiator caspases [5].
Molecular pathways leading to apoptosis are evolu-
tionarily conserved and are regulated by specific cellular
proteins. Some, such as Bcl-2, control the release of
proapoptotic proteins from the mitochondria [6].
Others, including various cellular inhibitor-of-apoptosis
proteins (IAPs), bind directly to active caspases and act
as natural inhibitors of caspase activity [7]. The IAP
family, currently identified in humans consists of
X-linked inhibitor of apoptosis protein (XIAP), ILP-2,

cIAP1, cIAP2, ML-IAP, NAIP, survivin, and livin
[7–9]. All IAPs have so-called conservative BIR
domains, which are responsible for their interaction with
caspases. XIAP is the most potent of all known IAPs
and contains three BIR domains. The third BIR domain
(BIR3) selectively targets caspase 9, whereas BIR2 and
the linker region between BIR1 and BIR2 inhibit effec-
tor caspases 3 and 7 [5,10]. This inhibition can be
relieved by IAP antagonists, which bind to IAPs pre-
venting caspase binding [5,8,9,11–13]. Recent studies
have revealed that binding of IAP antagonists to IAPs
may stimulate their auto-ubiquitination and degrada-
tion, thereby preventing caspase inhibition [14,15].
Recognition of XIAP as a direct inhibitor of caspases
makes it an attractive therapeutic target. This led to an
active search for any suitable molecular inhibitor
capable of easily penetrating a tumor cell to block
XIAP activity in the cytosol [8,9,16]. The discovery of
endogenous regulators of IAP activity enhanced these
investigations. Several intracellular inhibitory IAPs
have been characterized in humans, namely, Smac ⁄ DI-
ABLO, Omi ⁄ HtrA2, GSPT1⁄ eRF3, ARTS, and XAF1
[17–21]. However, only the first and best characterized
anti-IAP Smac ⁄ DIABLO is currently known to be
directly involved in the regulation of apoptosis. Other
anti-IAPs, such as Omi ⁄ HtrA2 or GSPT1 ⁄ eRF3, seem
to have a primary physiological role that is not directly
related to XIAP ⁄ caspase regulation [8,18,19]. Smac ⁄
DIABLO is released from mitochondria into the cyto-
sol during apoptosis, wherein it can bind to XIAP [17].

The main highly conserved functional motif common
to all IAP antagonists, was termed the IAP-binding
motif (IBM), and became a target for finding novel
potential inhibitors of IAP [8,9,16]. The motif ATPF ⁄
AVPI was first characterized in caspase 9 and
Smac ⁄ DIABLO was characterized as being responsible
for binding to BIR3 of XIAP [22].
Smac-derived peptides modeling their XIAP-binding
site, bind to recombinant BIR3 domains in vitro
[18–23]. Recent studies have set out to design small
molecular drugs carrying the IBMs [5,11–13] or artifi-
cial chimerical peptides composed of the IBM sequence
fused to a carrier peptide [23]. The cell-permeable
Smac peptides allowed the apoptosis resistance and
chemoresistance of cancer cells with a high level of
XIAP to be overcome in vitro and in vivo, as documen-
ted [13,23]. Despite the strong molecular basis for
interaction with XIAP, natural Smac-derived peptides
and other artificial IBM-based chimeric constructions
have several intrinsic limitations (e.g. poor in vivo
stability and very low bioavailability) making them
unsuitable for the treatment of cancer [8,9,23]. The
other known natural XIAP-binding proteins cannot
act as anticancer drugs because of their exclusive intra-
cellular location. Therefore, the search for other
XIAP-interacting and cell-membrane-penetrating drugs
is a highly desirable goal.
Recently, it was discovered that the well-known
oncofetal antigen a-fetoprotein (AFP) is able to induce
apoptosis selectively in tumor cells without any toxicity

towards normal cells and tissues [24–28]. AFP is one
of the major serum embryonic proteins involved in the
regulation of growth and the development of immature
embryonic tissues [29–31]. The specific expression and
internalization of AFP is restricted to developing cells,
such as embryonic cells, activated immune cells and
tumor cells, which suggests that it has an important
regulatory role in cell growth and differentiation [32–
35]. AFP expression is blocked completely after birth
and is recovered only after malignant transformation
[29–31]. Various researchers have documented the
existence of specific receptor-dependent mechanisms
responsible for the active endocytosis of AFP by
malignant cells [34–36]. AFP has been well character-
ized as a transport protein delivering natural ligands
such as fatty acids, hormones, and heavy metals to
developing cells [29]. The specific expression and inter-
nalization of AFP by developing cells, such as embry-
onic cells or tumor cells, together with the properties
of the transport protein make AFP very attractive for
tumor-targeting therapy [29,30,33]. The growth-regula-
tory activity of AFP and AFP derivatives has been
demonstrated by various authors [24–28,37–43]. Spe-
cial interest has focused on the tumor-suppressive
effects of AFP and its peptide derivatives [24–28,38,
39,42]. The growth-suppressive activity of AFP can be
realized by inducing apoptosis in many types of tumor
or activated immune cells [24–28,41]. AFP can trigger
apoptosis in tumor cells via activation of caspase 3,
independent of the membrane-receptor signaling [26].

AFP stimulates formation of the apoptosome complex,
and enhances recruitment and activation of caspases 3
AFP antagonizes XIAP function E. Dudich et al.
3838 FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS
and 9 by displacing cIAP-2 from the apoptosome
and from its complex with recombinant caspases 3 and
9 [28].
Based on the molecular mechanisms of AFP-medi-
ated apoptosis, we hypothesized that AFP might inter-
act with XIAP by displacing it from the complex with
caspases, and thus preventing caspase inhibition. We
demonstrate here that AFP physically associates with
XIAP in cytochrome c-activated cellular lysates, and
that this complex does not contain the effector
caspase 3. We found that purified human AFP binds
to recombinant XIAP, disrupts the association between
XIAP and activated caspase 3, and antagonizes the
antiapoptotic function of XIAP. Our data indicated
that AFP could also bind free XIAP to eliminate it
from the reaction area and prevent caspase binding.
Results
AFP promotes caspase activation in cell-free
cytosolic extracts by blocking of XIAP-dependent
inhibition
Recent evidence has shown that AFP promotes the
processing and activation of procaspase 3 in the pres-
ence of low suboptimal doses of cytochrome c in cell-
free cytosolic extracts. Simultaneously, AFP induced
the release of cIAP2 from the apoptosome complex
[28]. Our recent experimental data allowed us to hypo-

thesize that AFP could operate as a XIAP antagonist
by affecting the interaction of XIAP with active caspas-
es, thus promoting their activity. To determine whether
AFP can affect caspase 3 activity in HepG2 cytosolic
extracts in the presence of an inhibitory amount of exo-
genous XIAP, we monitored caspase activation in a
cell-free system. Cytosolic cell extracts were activated
by the addition of cytochrome c ⁄ dATP together with
AFP or human serum albumin (HSA) in the presence
of an inhibitory amount of rhXIAP. Figure 1 shows
that addition of XIAP induced 50% inhibition of
caspase 3 activity in activated cell-free extracts. Addi-
tion of AFP in the cytosolic extract induced significant
enhancement of the DEVD-ase activity. The same
caspase activity was detected upon the simultaneous
addition of an inhibitory amount of exogenous rhXIAP
together with AFP. The data clearly show that AFP
abrogated the inhibitory activity of exogenous rhXIAP
against endogenous caspases and failed to relieve
AFP-mediated caspase stimulation. By contrast, exo-
genous HSA did not affect caspase activity in cell-free
extracts (Fig. 1). Hence, AFP counteracts with XIAP
by abrogation its caspase inhibition in the cyto-
chrome c-activated cell-free cytosolic extracts.
AFP promotes caspase 3 activity exclusively
by abrogation of XIAP-dependent inhibition
To study direct AFP ⁄ XIAP ⁄ caspase 3 interaction we
used recombinant proteins to form a reaction mixture
in order to avoid the influence of other active com-
pounds, which are available in cytosolic extracts. An

effective amount of rhXIAP was added into the solu-
tion of active recombinant caspase 3 to induce 50%
decrease of its activity. The kinetics of the DEVD-ase
cleavage in the reaction mixture was monitored each
5 min intervals. rhXIAP in combination with HSA
(Fig. 2A) or alone (Fig. 2B), induced twofold inhibi-
tion of caspase 3 activity, but AFP ⁄ rhXIAP pretreat-
ment significantly reduced inhibition by rhXIAP
(Fig. 2A). Addition of AFP or HSA alone did not
affect caspase 3 activity (Fig. 2B). The results show
that AFP does not directly affect caspase 3 activity,
but targets XIAP by blocking its inhibitory activity
against caspase 3. Therefore, AFP antagonizes XIAP
function.
AFP competes with caspase 3 and caspase 9
for XIAP binding
Functional interference of AFP and XIAP to examine
their effect on caspase activity implied a direct physical
interaction. We further studied whether AFP can com-
pete with caspases 3 and 9 for XIAP binding. Pure
recombinant His-tagged active caspases 3 and 9 were
Fig. 1. AFP antagonizes XIAP-mediated caspase inhibition in cyto-
chrome c-activated cell-free cytosolic extracts. HepG2-derived
cytosolic extracts were activated by 1 m
M dATP and 5 lM cyto-
chrome c and incubated with or without rhXIAP (250 n
M) with the
addition of 400 n
M AFP or 400 nM HSA for 1 h at 30 °C. Control
lysates incubated without addition of HSA and AFP were taken as

controls. Caspase activity was measured by DEVD–AMC cleavage.
The mean data in RFU ± SD from three independent experiments
are shown.
E. Dudich et al. AFP antagonizes XIAP function
FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3839
incubated with rhXIAP and AFP or HSA, and protein
complexes were thereafter immobilized on Ni–Seph-
arose beads. After extensive washing the supernatant
and pellets (beads) were blotted and probed with anti-
bodies to XIAP. AFP, but not HSA, completely abro-
gated the association of rhXIAP with caspases 3 and 9
(Fig. 3, pellet). Western blotting revealed the presence
of XIAP in the supernatants from Ni resin treated
with AFP, but only a negligible amount of free
rhXIAP was detected in the supernatants of HSA-trea-
ted samples (Fig. 3). Western blotting of pellets using
antibodies against caspase 3 and caspase 9 demonstra-
ted that neither AFP nor HSA was able to modulate
binding of His-tagged caspases on the nickel resin
(Fig. 3). The data clearly demonstrated that AFP
cointeracted with XIAP by preventing XIAP ⁄ caspase
complex formation.
AFP coprecipitates with endogenous XIAP
in cellular extracts
We further studied the ability of AFP to interact with
endogenous XIAP in whole-cell extracts preactivated
with cytochrome c ⁄ dATP ⁄ AFP. Therefore, we exam-
ined whether AFP might be directly associated with
XIAP or ⁄ and caspase 3 in cell-free extracts. Protein
complexes were precipitated by the addition of corres-

ponding antibodies and protein A–Sepharose beads.
Complex formation was detected by immunoblotting
of the proteins bound to the protein A–Sepharose with
anti-XIAP, anti-AFP, or anti-(caspase 3) IgG. Figure 4
shows that AFP coprecipitated with endogenous XIAP
(Fig. 4A, lane 3) but not with caspase 3 (Fig. 4C, lane
2; C, lane 3), whereas XIAP coprecipitated with both
AFP and caspase 3 (Fig. 4A, lanes 2, 3). These results
show that AFP associates physically with endogenous
XIAP in activated cell cytosolic extracts (Fig. 4A,
Fig. 2. AFP abrogates XIAP-mediated inhibition of caspase 3 activity in vitro. Active recombinant caspase 3 (3 nM) was treated with: (A) a
mixture of rhXIAP (200 n
M) with AFP (400 nM) or HSA (400 nM); (B) AFP (400 nM), HSA (400 nM), XIAP (200 nM) or without additions.
Caspase 3 activity was measured by monitoring of DEVD–AMC cleavage at 5-min intervals. Data were collected at 30 °C for 30 min and
expressed in RFU. The mean ± SD of three independent determinations is shown.
Fig. 3. AFP prevents XIAP ⁄ caspases complex formation. Human
recombinant XIAP was incubated for 2 h at 4 °C with mixed
His-tagged active recombinant caspase 3 and caspase 9 in the
presence of AFP or HSA as described in Experimental procedures.
Protein complexes were precipitated by Ni–Sepharose beads.
Ni–Sepharose-bound proteins (pellet) and supernatants were ana-
lyzed by SDS ⁄ PAGE ⁄ immunoblotting with anti-XIAP, anti-(caspase 3)
and anti-(caspase 9) sera. Input 1: rhXIAP (100 ng); Input 2: recom-
binant His-tagged caspase 3 (50 ng); Input 3: recombinant His-
tagged caspase 9 (50 ng).
AFP antagonizes XIAP function E. Dudich et al.
3840 FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS
lane 3). As expected, endogenous caspase 3 coprecipi-
tated with endogenous XIAP (Fig. 4A, lane 2). No
interaction between caspase 3 and AFP could be detec-

ted (Fig. 4B, lane 2; Fig. 4C, lane 3). This indirectly
proved that AFP and caspase 3 interacted with the
same binding site of XIAP. If AFP had been attached
to a binding site on the XIAP molecule other than that
responsible for caspase 3 binding, we would be able to
detect coprecipitation of all three proteins in this
experiment. Hence, the results showed that AFP act-
ively binds to endogenous XIAP in cytochrome c-acti-
vated cellular extracts, but that it also prevents
complex formation of XIAP with active caspases. The
data also suggested that AFP seems to bind to the
entire XIAP molecule only, because fragmented XIAP,
which was available in the cytosolic extracts (Fig. 4A,
lane 1), was not recovered on the immunoprecipita-
tion ⁄ western blotting pattern with anti-(caspase 3) and
anti-AFP IgG within the limits of detection (Fig. 4A,
lanes 2, 3).
AFP physically associates with rhXIAP to form
high molecular mass complexes
We then determined whether AFP and XIAP were
able to form intermolecular complex. rhXIAP was
coincubated with rhAFP and then the protein mixture
was subjected to native electrophoresis. The complex
formation was analyzed by western blotting with anti-
AFP IgG (Fig. 5A, lanes 1, 2) and with anti-XIAP
IgG (Fig. 5B, lanes 1, 2). Probing with anti-AFP
revealed three AFP-specific bands (Fig. 5A, lane 2),
which correspond to the AFP-monomer, natural AFP-
dimer and high molecular mass upper band corres-
ponding to the AFP-specific macromolecular complex.

To identify presence of XIAP in the AFP-specific com-
plexes, we probed the blot pattern with anti-XIAP
IgG. This revealed presence of XIAP in the upper
band, corresponding to the high molecular mass AFP-
specific complex (Fig. 5B, lane 2). We conclude that
incubation of pure AFP with XIAP led to the forma-
tion an intermolecular complex. This AFP ⁄ XIAP
complex evidently contains more than two proteins,
showing the ability of AFP to form multimolecular
high-affinity complexes with XIAP.
A
B
C
Fig. 4. AFP associates physically with endogenous XIAP in the cel-
lular cytosolic extracts. HepG2 cytosolic extract was activated by
the addition of 5 l
M cytochrome c and 1 mM dATP for 30 min at
30 °C in the presence of AFP (6 lg) and thereafter the specific
interaction of AFP, XIAP and caspase 3 was tested using coimmu-
noprecipitation with anti-AFP, anti-(caspase 3) or anti-(rabbit IgG) as
negative control. Western blot analysis was carried out with anti-
XIAP (A), anti-AFP (B) and anti-(caspase 3) (C) sera. Input: cytosolic
extract activated with cytochrome c ⁄ dATP (20 lg). Molecular mass
markers are indicated on the left.
AB
Fig. 5. Direct AFP ⁄ XIAP complex formation in vitro. Recombinant
human AFP and XIAP were coincubated for 1 h at 4 °C and there-
after subjected to the native nondenaturing PAGE followed by
western blotting with anti-AFP (A) and anti-XIAP (B) IgG. Lane 1 on
the each pattern corresponds to rhAFP alone and lane 2 corres-

ponds to AFP ⁄ XIAP.
E. Dudich et al. AFP antagonizes XIAP function
FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3841
Search for the potential IBM in the structure of
the AFP molecule
The AFP protein contains a putative IBM-like
sequence ATIF(29–32), which fits the IAP binding
tetrapeptide consensus [9,44] (Fig. 6). Similar to the
other IBM proteins, Smac, Omi and GSPT1 [17–19],
AFP requires N-terminal processing to expose the
IBM motif at the newly generated N-terminus.
Processing of the first 28 amino acids could allow
exposing of N-terminal motif ATIF that is highly
reminiscent of IBM of caspase 9 and other IAP antag-
onists (Fig. 6). In common with other IBMs, the IBM-
like motif of AFP bears Ala at its N-termini. The Ala
residue within the IBM is highly conserved (Fig. 6)
and has been shown to be essential for the interaction
between XIAP and mature Smac⁄ Diablo [45]. This
sequence displays a high degree of similarity to the
IBM of caspase 9 with a single replacement of Pro3 in
caspase 9 to Ile3 in AFP (Fig. 6) [22]. This position is
variable in different IAP antagonists and does not
seem to be critical in forming the XIAP-binding site
(Fig. 6). The AFP IBM-like sequence has Phe at the
P5 position, as in Drosophila Sickle and Grim and
Xenopsis Casp-9 (Fig. 6) [22,46]. As shown previously
[46], Phe at P5 position is clearly favored for BIR2
binding. The requirement for proteolytical processing
of AFP to expose the N-terminal IBM may explain

why only part of the total amount of AFP, that which
has undergone proteolytical processing, participates in
complex formation with XIAP (Fig. 5A). Proteolytical
processing of AFP is usually observed in cyto-
chrome c-activated lysates [28]. It has been shown that
proteolytical cleavage of pure AFP results in AFP
fragments exposing different destabilizing N-terminal
residues [27]. However, our results show that pure
recombinant AFP and XIAP can interact without any
requirements for the presence of active caspases in the
reaction mixture. We tentatively suggest the existence
of another IAP-binding site in AFP, one which does
not require N-terminal processing to be activated for
XIAP binding.
Structural modeling of the AFP(dimer)
⁄
BIR2–3
complex
Our results show that AFP can bind to an entire XIAP
molecule but not to its fragments. It has also been
demonstrated that AFP displaces caspase 3 from its
complex with XIAP, suggesting that the BIR2 domain
is involved in this interaction. The data also suggest
that AFP uses at least two different XIAP binding
sites to form the AFP–XIAP complex. Previous studies
suggest that AFP [27], as well as XIAP [47], is able to
Fig. 6. Sequence alignment of IBM-bearing proteins. Collinear alignment of the N-terminal sequence 1–55 from HSA and 1–60 from human
AFP (upper). Sequence alignment of IBM-bearing proteins: human AFP, caspase 9–p12, caspase 7–p20, Smac ⁄ DIABLO, GSPT1; Omi ⁄ Htr2;
mouse caspase 9–p12; Xenopus caspase 9–p12; Drosophila ICE, Reaper, Grim, Hid, Sickle, Jafrac2, GSPT1; C. elegans GSPT1. Identical res-
idues are highlighted in black. Residues conserved in several IBM proteins are indicated in grey. IBM-like sequence is boxed. Protein

sequence data have been taken from the Protein Data Bank [63].
AFP antagonizes XIAP function E. Dudich et al.
3842 FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS
dimerize, which could create many possibilities for
interaction stoichiometry. We suggest that AFP dimer
forms a complex with XIAP by interacting with both
BIR2 and BIR3 domains. The involvement of BIR3 in
the interaction with AFP is supported by recent studies
showing that AFP induced the release of both
caspase 3 and caspase 9, as well as cIAP-2, from the
apoptosome complex [28]. It is possible that caspase 9
cointeracts with AFP⁄ XIAP complex similarly to
caspase 3. It is expected that AFP dimer interacts with
the BIR2 and BIR3 domains of XIAP and forms a
2 : 1 stoichiometric complex. Native electrophoresis
indicates that the AFP ⁄ XIAP complex is formed by
more than two molecules and includes at least three
members (Fig. 5). Taking into account that both AFP
and XIAP tend to dimerize, a few interaction models
can be proposed. We suggest a simple complex com-
posed of AFP dimer and XIAP monomer.
The 3D molecular structure of the AFP molecule
remains unsolved. Human AFP and HSA exhibit 39%
amino acid sequence homology [44]. The authentic
structural homology of AFP and HSA allowed us to
predict the tertiary structure of AFP based on the
atomic coordinates obtained by X-ray crystallography
for HSA [48]. Using the NMR structures of the BIR2
[49] and BIR3 [50] domains, the AFP(dimer) ⁄ BIR2–3
complex was constructed (Fig. 7). In this complex, the

BIR2 and BIR3 domains show the same local twofold
symmetry as two AFP monomers in the AFP dimeric
structure. Moreover, the IBM-interacting grooves of
the BIR2 and BIR3 domains lie close to the ATIF-end
of the first and second AFP monomers, respectively,
allowing for the possibility that they belong to the
same XIAP molecule.
Discussion
Recent evidence has broken the main paradigm of
apoptosis, stating that the release of cytochrome c is
the point of no return in the apoptotic program
[17,18,51,52]. It has been shown that certain tumor
cells are able to recover after cytochrome c release and
survive despite the constitutive presence of cyto-
chrome c in the cytosol in the absence of any signs of
apoptosis [53]. Moreover, caspase activation does not
always result in cell death [16–18,52]. The ability of
the cell to die at the postmitochondrial level depends
mainly on the activity of endogenous inhibitors of
apoptosis, such as IAPs, sHSPs, or Bcl-2 [6,7,54,55].
There is further evidence of a high level of apoptotic
activation and the upregulation of IAPs in tumor tis-
sue [8,16]. Inactivation of XIAP or the cancellation of
XIAP inhibition appears both necessary and sufficient
for cytochrome c to activate caspases and trigger cell
death [9,16]. The activity of IAPs is regulated by a
group of IAP-regulatory proteins that bind to IAPs
and inhibit their antiapoptotic function [17–21]. These
factors are important research targets to search for
new nontoxic drugs with selective pro-apoptotic activ-

ity for tumor cells. The identification of protein
drugs, which can overcome the tumor defense system
by preventing the realization of apoptosis in tumor
cells, will have a great potential as tumor therapeutic
agents [9].
In this study we showed that AFP could participate
in the regulation of apoptosis in tumor cells by coun-
teraction with the most potent endogenous inhibitor of
mammalian caspases XIAP. Our results show that
AFP binds to XIAP and disrupts its interaction with
caspase 3. These results are in harmony with the fact
that AFP can bind to cIAP-2 and disrupt its interac-
tion with caspase 3 and caspase 9 [28]. Because pure
AFP can bind XIAP in vitro, this interaction appears
to be direct. The binding seems to be highly specific,
because it did not occur with the nonapoptotic protein
HSA, structure and function of which is closely related
to AFP. In addition to the direct association with
XIAP, AFP could also relive the XIAP-inhibitory
effect on the activity of the mature recombinant
caspase 3. Moreover, AFP shows the ability to enhance
Fig. 7. Hypothetical molecular model of the AFP(dimer) ⁄ BIR2–3
complex. Each monomer of the AFP dimer is shown in blue and
red, respectively. The BIR2 and BIR3 domains are shown in green.
The dashed yellow lines connect the ATIF peptides of each AFP
monomer and the IBM-interacting grooves of the BIR2 and BIR3
domains. The figure was produced using
MOLSCRIPT v. 2.1 [62].
E. Dudich et al. AFP antagonizes XIAP function
FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3843

cytochrome c-dependent activation of caspase 9 and
caspase 3 in the presence of an inhibitory amount of
exogenous XIAP. In this respect, AFP behaves in a
similar manner to the IAP antagonists. This group of
proteins is characterized by the presence of the N-ter-
minal conserved BIR-binding motif (IBM), which is
required for IAP binding [20]. The presence of IBM in
the intracellular protein allowed us to recognize its
possibility of serving as an IAP antagonist [9,13,23].
Mammalian proteins Smac ⁄ DIABLO, Omi ⁄ HtrA2
and GSPT1 ⁄ eRF3 are released from mitochondria
upon triggering apoptosis and require processing to
reveal the IBM at the newly generated N-terminus
[17–19]. However, other proteins, such as ARTS and
XAF1, which do not contain an IBM-like motif, were
also seen to antagonize IAP function via an unknown
mechanism [20,21]. The search for the potential IBM-
like sequence in the structure of AFP revealed the
presence of a similar amino acid sequence ATIF in
human AFP at position 29–32 [44]. It can be proposed
that processing of the first 28 amino acid residues gen-
erates the AFP fragment with an N-terminal motif
ATIF that is highly reminiscent of IBM of caspase 9
and other IAP antagonists (Fig. 6).
Our results indicate that only entire XIAP could
bind AFP (Fig. 5). This means that AFP binds simul-
taneously to at least two BIR domains. BIR3 binding
is preferential for an IBM-like motif similar to that
available in caspase 9. Taking into account that AFP
competes with caspase 3 for complex formation with

XIAP, we consider that both BIR2 and BIR3 may be
involved in complex formation with AFP. A similar
model has been described previously [56] for a complex
of dimeric Smac protein with recombinant XIAP frag-
ments containing both BIR2 and BIR3 domains, or
for a complex of XIAP with active caspases 3 and -7
[10]. Another model, which involves the entire AFP
molecule, could be also proposed, and will introduce
other parts of the molecule in their interaction with
XIAP. To identify the molecular mechanism of the
AFP ⁄ XIAP interaction additional structural studies
are needed.
Although our results show that AFP interacts phys-
ically with XIAP and protects activated caspases from
IAP-induced inhibition, they do not reveal how it
operates. There are several possibilities. However, a
functional preference of AFP for tumor cells seems evi-
dent [30–41]. It has previously been shown that AFP
selectively penetrates tumor cells via specific membrane
AFP receptors expressed on the surface of tumor cells
but not on normal adult cells [32–36]. Unlike other
anti-IAPs, such as Smac or Omi, which have an exclu-
sively mitochondrial localization and become available
to interact with IAPs only after apoptosis has been
triggered by cytochrome c release, AFP was available
whenever it entered into the cell via cellular membrane
receptors or was synthesized inside the cell. Thus, AFP
is able to regulate the IAP level in the cytosol inde-
pendently of whether cell is undergoing apoptosis or
not. Under conditions of constitutively high levels of

XIAP expression in tumor cells [57], AFP could reduce
its protein level, presumably by proteosome-mediated
degradation.
Comparison with normal cell lines and tissues has
shown that many tumor cell lines and tissues have
constitutively higher levels of active caspase and free
cytochrome c in the absence of apoptotic stimuli and
yet are not undergoing apoptosis [58,59]. Simulta-
neously, tumor cells have high levels of expression of
survivin and XIAP [57,60]. Survival of cancer cells is
possible under conditions of pacific equilibrium
between pro- and antiapoptotic signals. Taking into
account that normal cells and tissues do not overex-
press apoptotic stimuli and IAPs, whereas cancer cells
and tissues do, IAP-targeting drugs will have highly
selective proapoptotic activity for cancer cells and lit-
tle toxicity towards normal cells [8,9,16]. The general
obstacle preventing the design of apoptosis-regulating
drugs on the basis of known natural anti-XIAPs is
their intracellular localization and the inability to use
them as internal regulating factors. Considering that
AFP can penetrate selectively into tumor cells via
specific membrane receptors, the molecular mechan-
ism of AFP-mediated targeting regulation of apopto-
sis could be suggested to be as follows: (a) AFP
selectively penetrates tumor cells via specific AFP re-
ceptors; and (b) formation of the AFP–XIAP com-
plex prevents its binding to activated caspases,
increases XIAP instability against ubiquition ⁄ protea-
somal destruction and reduces the XIAP level to pro-

mote apoptosis induction. This function of AFP may
serve to sensitize tumor cells to weak proapoptotic
stimuli by inducing a tumor-specific response to che-
motherapeutic or radiotherapeutic treatments. The
selectivity of the AFP-mediated proapoptotic activity
for tumor cells may be explained by its counteraction
with IAPs, which are shown to be dominantly over-
expressed in tumor cells under conditions of the sim-
ultaneous existence of high levels of various active
proapoptotic factors.
Normal cells do not undergo AFP-induced apoptosis
because they do not express high levels of IAPs, do
not contain constitutively activated caspases and do
not express membrane AFP receptors. AFP seems to
be directly involved in targeting positive regulation of
the apoptotic pathway dysfunction in cancer cells by
AFP antagonizes XIAP function E. Dudich et al.
3844 FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS
inhibition of IAP function leading to triggering of the
apoptosis machinery.
Further studies are required to better understand the
importance of the role AFP in modulating the level of
IAPs in tumor cells. Elucidation of the role of AFP
in tumor cell-specific regulation of XIAP function in
apoptosis may have important implications for cancer
treatment and prevention. AFP and AFP-derived pep-
tides can potentially be used to overcome drug resist-
ance caused by the differential mechanism of apoptosis
dysfunction in cancer cells.
Experimental procedures

Purification of a-fetoprotein
Embryonic AFP was isolated from human cord serum
using ion-exchange, affinity and gel-filtration chromatogra-
phy as described previously [27]. The purity and homogen-
eity of the protein were assessed by SDS ⁄ PAGE and
western blotting with AFP-specific polyclonal antibodies
as described elsewhere [28]. Recombinant human AFP
(rhAFP) was purified from the culture medium of recom-
binant Saccharomyces cerevisiae as described previously [43]
using affinity and gel chromatography.
Cells
HepG2 cells originating from the American Type Culture
Collection were grown in Dulbecco’s modified Eagle’s med-
ium (ICN Biomedicals, Inc., Costa Mesa, CA) with addition
of l-glutamine, 10% heat-inactivated fetal bovine serum,
penicillin (100 unitsÆmL
)1
), streptomycin (0.1 mgÆmL
)1
)ina
humidified 5% CO
2
atmosphere at 37 °C. For a passage,
cells were incubated in 0.25% trypsin solution, then washed
and plated out.
Preparation of cell-free cytosolic extracts
Cell-free cytosolic extracts were generated from human
hepatocarcinoma HepG2 as described previously [60] with
minor modifications [28]. Cells (4 · 10
8

) were collected and
washed three times with 50 mL NaCl ⁄ P
i
and once with
5 mL hypotonic cell extraction buffer (CEB; containing
20 mm Hepes, pH 7.2, 10 mm KCl, 2 mm MgCl
2,
1mm
dithiothreitol, 5 mm EGTA, 2 5 lgÆmL
)1
leupeptin, 5 lgÆmL
)1
pepstatin, 40 mm b-glycerophosphate, 1 mm phenyl-
methylsulfonyl fluoride). The cell pellet was then resuspend-
ed in an equal volume of CEB, allowed to swell for 20 min
on ice and then disrupted by passing through a needle. The
homogenate was centrifuged at 5000 g for 10 min at 4 °C
to remove whole cells and nuclei. Thereafter the superna-
tant was centrifuged at 15 000 g for 20 min at 4 °C. The
procedure was repeated twice. Cytosolic extracts were
assessed for protein content by Bradford assay and stored
in aliquots at )70 ° C.
Analysis of caspase activity
Caspase assays were performed with active recombinant
caspase-3 (Alexis Corp., San Diego, CA), recombinant full-
length rhXIAP (R&D Systems, Minneapolis, MN), purified
AFP, and HSA (Sigma-Aldrich, St. Louis, MO). All other
reagents were from Sigma, unless stated otherwise.
RhXIAP (200 nm) was incubated with AFP (400 nm)or
HSA (400 nm) in IAP buffer (50 mm Hepes, pH 7.5,

100 mm NaCl, 1 mm EDTA, 5 mm dithiothreitol, 0.1%
Chaps, 10% sucrose) for 15 min at room temperature.
Thereafter the active recombinant caspase 3 (3 nm) was
added to the reaction mixture, and incubation continued
for a further 15 min under the same conditions. For the
control, caspase 3 was incubated with each of the following
compounds separately: AFP (400 nm), HSA (400 nm)or
XIAP (200 nm). The kinetics of caspase activity was moni-
tored by cleavage of the fluorogeneic substrate [50 lm
Ac-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin (Ac-DEVD-
AMC), Sigma] at 5-min intervals for 30 min. To assess the
effects of AFP, HSA and XIAP on caspase activity in cellu-
lar extracts in vitro, rhXIAP (250 nm) was incubated with
cytosolic extract (40 lg) activated by addition of 5 lm of
bovine heart cytochrome c and 1 mm dATP in the presence
of AFP (400 nm) or HSA (400 nm)in15lL of a reaction
buffer (10 mm Hepes, pH 7.2, 25 mm NaCl, 2 mm MgCl
2
,
5mm dithiothreitol, 5 mm EDTA, 0.1 mm phenylmethyl-
sulfonyl fluoride) for 1 h at 30 °C, and the reaction
mixtures were analyzed for Ac-DEVD-AMC cleavage.
Caspase 3 activity was determined by adding 5 lL of cell
extracts to 16 lL of substrate reaction buffer (20 mm
Hepes, pH 7.2, 100 mm NaCl, 10 mm dithiothreitol, 1 mm
EDTA, 0.1% Chaps 10% sucrose, 50 lm Ac-DEVD-AMC)
for 40 min at 30 °C. The reaction was stopped by the addi-
tion 200 lL of cold NaCl ⁄ P
i
, and AMC liberation was

measured using Victor-1420 Multilabel counter (Wallac,
Finland) at excitation 355 nm and emission 460 nm. All
samples were analyzed in duplicate and the experiments
were repeated three times. For each sample, caspase activity
was expressed in relative fluorescent units (RFU), showing
the amount of cleaved substrate normalized for protein
content.
Direct protein–protein binding assay
To determine possible interactions between AFP, caspase 3,
caspase 9, and XIAP, we used a direct coprecipitation assay
with purified proteins. Human recombinant XIAP (350 ng),
His-tagged human recombinant caspase 9 (50 ng), anf act-
ive His-tagged rat recombinant caspase 3 (300 ng) were
mixed with 0.5 mgÆmL
)1
AFP or 0.5 mgÆmL
)1
HSA in
E. Dudich et al. AFP antagonizes XIAP function
FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3845
15 lL buffer A (20 mm Hepes, pH 7.4, 100 mm NaCl,
0.5 mm EDTA, 0.5 mm dithiothreitol, 0,1% Chaps, 10%
sucrose) and incubated for 2 h at 4 °C. Thereafter, 15 lL
of Ni-Sepharose beads (Qiagen, Valencia, CA) in 80 lLof
buffer B (20 mm Na
2
HPO
4
, pH 7.2, 0.2 m NaCl) were
added to the reaction mixture and incubation was contin-

ued for the next 1 h. The beads were separated from
supernatants by centrifugation and both fractions were
collected. Protein–bead complexes were washed four times
and boiled in 20 lL of reducing 2 · Laemmli sample buf-
fer. Samples, protein–beads and supernatants were analyzed
by SDS ⁄ PAGE ⁄ immunoblotting in 12.5% polyacrylamide
gel in with b-mercaptoetanol. To study direct AFP–XIAP
protein interaction, human recombinant XIAP (1.5 lg) and
human recombinant AFP (2 lg) were incubated for 1 h at
4 °Cin30lL of buffer C (20 mm Hepes, pH 7.2, 140 mm
KCl, 5 mm MgCl
2
,2mm dithiothreitol, 1 mm EDTA,
0.1% OVA). Thereafter, protein mixtures (5 lL) were sub-
jected to 8% nondenaturing continuous polyacrylamide gel
(Tris pH 8.7) and separated by Native electrophoresis [61].
Immunoprecipitation
Cytosolic extracts obtained from HepG2 cells were nor-
malized for protein content (500 lg of total protein in
100 lL of buffer C) and activated by addition of 5 lm
cytochrome c and 1 mm dATP for 30 min at 30 °C in the
presence of AFP (6 lg). The reaction mixtures were
cooled and incubated with 3 lg of the following antibod-
ies: polyclonal rabbit anti-AFP [28], normal rabbit IgG
(Sigma), or with rabbit anti-(caspase 3) (Santa Cruz, Santa
Cruz, CA) for 2 h with a gentle mixing at 4 °C. Thereaf-
ter, 40 lL of protein A–Sepharose bead slurry (Amersham
Pharmacia Biotech) were added to the each sample. Sam-
ples were incubated overnight in a rotating shaker at
4 °C. The beads were pelleted by centrifugation and after

intensive washing, were syringe dried. The bound proteins
were eluted by boiling in 25 lLof2· sample buffer. Sam-
ples in aliquots of 10 lL were loaded onto the 12.5%
SDS polyacrylamide gel and subjected to SDS ⁄ PAGE ⁄
immunoblotting.
Immunoblotting analysis
Samples after SDS ⁄ PAGE or native PAGE were electro-
blotted onto a poly(vinylidene difluoride) membranes
(Amersham Pharmacia Biotech) using a semidry transfer
apparatus (Bio-Rad Laboratories, Hercules, CA). Follow-
ing blocking, the membranes were incubated with primary
rabbit anti-AFP IgG [28] or goat anti-XIAP IgG (R&D
Systems, Minneapolis, MN), or anti-(caspase 3) IgG (Santa
Cruz) and then incubated with donkey anti-(rabbit IgG)
(Amersham Pharmacia Biotech) or anti-(goat IgG) (Imtek,
Russia) each conjugated to horseradish peroxidase. The
blots were visualized using ECL or ECL Plus method
(Amersham Pharmacia Biotech) according to manufacturer
instructions.
Theoretical calculation of protein structure
In order to predict the tertiary structure of the AFP mole-
cule [44] the molecular modeling software package sybyl
was used (Tripos Associates, Inc., St. Louis, MO). A model
of the dimeric structure of the AFP was reconstructed by
using of the atomic coordinates obtained by X-ray crystal-
lography for HSA [48] and plotted using molscript v. 2.1
[62]. The atomic coordinates of HSA (code 1AO6) were
obtained from the Protein Data Bank [63]. The primary
structure alignment of AFP and HSA was constructed
using multalin [64]. The model of AFP was minimized to

an energy gradient < 0.050 kcalÆmol
)1
ÆA
˚
)1
using the Tripos
force field and a combination of Simplex [65] and Powell
algorithms [66]. Coordinates of the NMR structures of the
BIR2 [49] and BIR3 [50] domains were achieved from the
Protein Data Bank files: 1C9Q and 1F9X, respectively.
Acknowledgements
This work was supported in part by the International
Science & Technology Center, ISTC (grant #1878); by
the Academy of Finland (Grant # 107762), the Neobi-
ology program of the Technology Development Center
for Finland (TEKES), and the Sigrid Juse
´
lius Founda-
tion.
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