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

Alpha-fetoprotein antagonizes X-linked inhibitor of apoptosis protein anticaspase activity and disrupts XIAP–caspase interaction Elena Dudich1,2, Lidia Semenkova1,2, Igor Dudich1,2, Alexander Denesyuk3, Edward Tatulov2 and Timo Korpela4

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

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: elena_dudich@mail.ru

(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.

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

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.

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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].

IAPs have

location. Therefore,

[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 intrinsic limitations (e.g. poor in vivo have several 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 the search for other XIAP-interacting and cell-membrane-penetrating drugs is a highly desirable goal.

Recently,

it was discovered that

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 from the mitochondria [6]. proapoptotic proteins 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 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].

intracellular

characterized in caspase 9

Recognition of XIAP as a direct inhibitor of caspases makes it an attractive therapeutic target. This led to an inhibitor active search for any suitable molecular 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 inhibitory IAPs investigations. Several 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 and Smac ⁄ DIABLO was characterized as being responsible for binding to BIR3 of XIAP [22].

Smac-derived peptides modeling their XIAP-binding in vitro

site, bind to recombinant BIR3 domains

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

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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

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 mM dATP and 5 lM cyto- chrome c and incubated with or without rhXIAP (250 nM) with the addition of 400 nM AFP or 400 nM HSA for 1 h at 30 (cid:2)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.

AFP promotes caspase activation in cell-free cytosolic extracts by blocking of XIAP-dependent inhibition

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- significantly reduced inhibition by rhXIAP ment (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

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 the DEVD-ase activity. The same enhancement of 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.

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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 nM) 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 (cid:2)C for 30 min and expressed in RFU. The mean ± SD of three independent determinations is shown.

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

Input 3:

Fig. 3. AFP prevents XIAP ⁄ caspases complex formation. Human recombinant XIAP was incubated for 2 h at 4 (cid:2)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- recombinant His- binant His-tagged caspase 3 (50 ng); tagged caspase 9 (50 ng).

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,

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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

A

B

C

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- lanes 1, 2) and with anti-XIAP AFP IgG (Fig. 5A, lanes 1, 2). Probing with anti-AFP IgG (Fig. 5B, lane 2), revealed three AFP-specific bands (Fig. 5A, 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

Fig. 4. AFP associates physically with endogenous XIAP in the cel- lular cytosolic extracts. HepG2 cytosolic extract was activated by the addition of 5 lM cytochrome c and 1 mM dATP for 30 min at 30 (cid:2)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.

that

Fig. 5. Direct AFP ⁄ XIAP complex formation in vitro. Recombinant human AFP and XIAP were coincubated for 1 h at 4 (cid:2)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.

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 three proteins in this detect coprecipitation of all experiment. Hence, the results showed that AFP act- ively binds to endogenous XIAP in cytochrome c-acti- vated cellular extracts, but 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,

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Search for the potential IBM in the structure of the AFP molecule

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

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 the newly generated N-terminus. IBM motif at 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

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].

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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

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

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].

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].

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

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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.

conditions of pacific

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.

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 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.

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

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

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assessed for protein content by Bradford assay and stored in aliquots at )70 (cid:2)C.

inhibition of IAP function leading to triggering of the apoptosis machinery.

Analysis of caspase activity

from Sigma, unless

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.

in IAP buffer

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) in a humidified 5% CO2 atmosphere at 37 (cid:2)C. For a passage, cells were incubated in 0.25% trypsin solution, then washed and plated out.

Preparation of cell-free cytosolic extracts

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 stated otherwise. RhXIAP (200 nm) was incubated with AFP (400 nm) or HSA (400 nm) (50 mm Hepes, pH 7.5, 100 mm NaCl, 1 mm EDTA, 5 mm dithiothreitol, 0.1% for 15 min at room temperature. Chaps, 10% sucrose) 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- the fluorogeneic substrate [50 lm tored by cleavage of 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) in 15 lL of a reaction buffer (10 mm Hepes, pH 7.2, 25 mm NaCl, 2 mm MgCl2, 5 mm dithiothreitol, 5 mm EDTA, 0.1 mm phenylmethyl- for 1 h at 30 (cid:2)C, and the reaction sulfonyl fluoride) 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 (cid:2)C. The reaction was stopped by the addi- tion 200 lL of cold NaCl ⁄ Pi, 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

40 mm b-glycerophosphate,

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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 Cell-free cytosolic extracts were generated from human hepatocarcinoma HepG2 as described previously [60] with minor modifications [28]. Cells (4 · 108) were collected and washed three times with 50 mL NaCl ⁄ Pi and once with 5 mL hypotonic cell extraction buffer (CEB; containing 20 mm Hepes, pH 7.2, 10 mm KCl, 2 mm MgCl2, 1 mm dithiothreitol, 5 mm EGTA, 25 lgÆmL)1 leupeptin, 5 lgÆmL)1 1 mm phenyl- pepstatin, 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 (cid:2)C to remove whole cells and nuclei. Thereafter the superna- tant was centrifuged at 15 000 g for 20 min at 4 (cid:2)C. The repeated twice. Cytosolic extracts were procedure was

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AFP antagonizes XIAP function

(Amersham Pharmacia Biotech) according to manufacturer instructions.

Theoretical calculation of protein structure

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 (cid:2)C. Thereafter, 15 lL of Ni-Sepharose beads (Qiagen, Valencia, CA) in 80 lL of buffer B (20 mm Na2HPO4, 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 (cid:2)C in 30 lL of buffer C (20 mm Hepes, pH 7.2, 140 mm KCl, 5 mm MgCl2, 2 mm 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]. 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)1ÆA˚ )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.

Immunoprecipitation

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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