Original Paper
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file on Synergy OPEN |
Acta Biochim Biophys
Sin 2008, 40: 278-288
doi:10.1111/j.1745-7270.2008.00407.x
Inhibitor of apoptosis
proteins and apoptosis
Yunbo Wei, Tingjun Fan*, and
Miaomiao Yu
Department of
Marine Biology, College of Marine Life Sciences, Ocean University of China,
Qingdao 266003, China
Received: December 11,
2007 Accepted: January
27, 2008
This work was supported
by a grant from the National High Technology Research and Development Program
of China (863 Program) (No. 2006AA10A401)
*Corresponding
author: Tel, 86-532-82031637; Fax, 86-532-82031637; E-mail, [email protected]
Apoptosis is a
physiological cell death process that plays a critical role in development,
homeostasis, and immune defense of multicellular animals. Inhibitor of
apoptosis proteins (IAPs) constitute a family of proteins that possess between
one and three baculovirus IAP repeats. Some of them also have a really
interesting new gene finger domain, and can prevent cell death by binding and
inhibiting active caspases, but are regulated by IAP antagonists. Some evidence
also indicates that IAP can modulate the cell cycle and signal transduction.
The three main factors, IAPs, IAP antagonists, and caspases, are involved in
regulating the progress of apoptosis in many species. Many studies and
assumptions have been focused on the anfractuous interactions between these
three main factors to explore their real functional model in order to develop
potential anticancer drugs. In this review, we describe the classification,
molecular structures, and properties of IAPs and discuss the mechanisms of
apoptosis. We also discuss the promising significance of clinical applications
of IAPs in the diagnosis and treatment of malignancy.
Keywords inhibitor
of apoptosis proteins; apoptosis; baculovirus IAP repeat; IAP antagonist
Apoptosis, a crucial biological process, plays an essential role in
regulating development, homeostasis, and immune defense by clearing redundant
or abnormal cells in organisms. A delicate balance between pro-apoptotic and
anti-apoptotic mechanisms determines whether a cell death signal can activate
the execution of the apoptotic program. In this balance, pro-apoptotic proteins
promote apoptosis and anti-apoptotic proteins inhibit apoptosis. As members of
the anti-apoptotic family of proteins, inhibitors of apoptosis proteins (IAPs)
can inhibit the downstream components of the caspase activation pathways in the
regulation of apoptosis and play important roles in regulating the progress of
apoptosis in many species [1,2].
IAP family members
The IAP gene was first identified in insect SF-21 cells
infected by baculovirus [3]. Encoded by a viral gene, this novel IAP can
inhibit infected SF-21 cells from executing apoptosis. It has similar
anti-apoptotic functions to p35 from Autographa californica multicapsid
nucleopolyhedrovirus but shows no significant homology. This finding indicated
that the IAP gene is 1.6 kb in size encoding a 31 kDa protein with a
zinc finger-like motif. Subsequent studies identified anti-apoptotic proteins that
can be grouped into the IAP family based on the presence of between one and
three baculovirus IAP repeats (BIR) domains at the N-terminus. Some IAPs also
have a really interesting new gene (RING) finger domain at the C-terminus. Many
IAP family members have been identified in diverse species ranging from viruses
to mammals (Table 1). In addition, eight human IAPs and three Drosophila
IAPs have been studied extensively. Some newly identified IAPs or IAP-like
proteins (ILPs) in new species have expanded the IAP family. In 2005, two
novel ILPs, AtILP 1 and AtILP 2, were identified in plant Arabidopsis
thaliana for the first time [4]. It was found that AtILPs have two
conserved BIR-like domains, as in human ILP-1, that might play some roles in
apoptosis. In Xenopus egg extracts, four maternal BIR family proteins
have recently been identified [5]. The survivin-related Xenopus
embryonic IAP, xEIAP/XLX, is inferior in apoptosis inhibition whereas xXIAP, a
possible ortholog of X-chromosome-linked IAP (XIAP), greatly delays apoptotic
initiation and is important for the survival of Xenopus eggs.
Molecular structures of IAP family members
IAP family members are characterized by the BIR domain, the name of
which derives from the original discovery of these apoptosis suppressors in the
genome of baculoviruses [3]. The BIR domains consist of approximately 70 amino
acids that contain the characteristic sequence CX2CX16HX6C. With both hydrophobic and hydrophilic
residues on its surface, the BIR core is theoretically capable of supporting
protein-protein interactions. There are three subtypes of BIR domain, BIR1,
BIR2, and BIR3, classified by their evolutionary relationship in phylogenesis.
All the molecular structures of IAP family members are shown in Fig. 1.The RING finger domain (C3HC4) exists at the C-terminal in some IAPs. It contains one zinc atom
chelated to three cysteines and one histidine and another zinc atom bound to
four cysteines. Some IAP family members also contain other structures, such as
the caspase activation recruitment domain, phosphate-loop and
ubiquitin-conjugating (UBC). Although the BIR domain is required for the
anti-apoptotic functions of the IAP family proteins, not all BIR-containing
proteins have anti-apoptotic functions.All the IAPs are homologs with highly conserved sequences. The close
relationship between baculoviral IAPs and insect IAPs suggests that baculoviral
IAPs might have been acquired through gene transfer from host insect cells.
Some baculoviral IAPs can even suppress apoptosis in mammalian cells [6].
Regulatory mechanism of IAP in apoptosis
The progress of apoptosis is regulated in an orderly way by a series
of signal cascades under certain circumstances. Three main factors, IAP, IAP
antagonist, and caspase, are involved in regulating this progress. The detailed
regulatory mechanism of IAP and the complicated interaction of the three main
apoptotic factors are shown in Fig. 2.
IAP as inhibitor of caspase
IAP acts as endogenous inhibitor of caspases, the main executioners of
apoptosis [7]. There are four pathways that have been described for caspase
activation in apoptosis initiation: (1) the mitochondrial pathway (intrinsic
pathway) where the release of cytochrome c from the mitochondria and the
formation of apoptosomes activate caspase-9 and in turn caspase-3; (2) the
death receptor pathway (extrinsic pathway), initiated by the ligand binding of
extracellular signals and death receptors FasL (Fas ligand)/Fas, tumor necrosis
factor (TNF)/TNF receptor on cell membrane; (3) the endoplasmic reticulum (ER)
stress-induced apoptosis pathway that leads to the activation of caspase-2 and
caspase-9; and (4) the activation by granzyme B of effector caspases by
injecting cytolytic T cells and natural killer cells to target cells. All of
these pathways converge on the activation of caspases, such as caspases 3, 6,
7, and 9. Four of the pathways are not distinct, in that the activation of one
usually involves another.Recent studies showed that IAP can inhibit the activity of caspases
by binding of their conserved BIR domains to the active sites of caspases in
vitro and in vivo. IAPs inhibit caspases by promoting the
degradation of active caspases, or by sequestering the caspases away from their
substrates [8]. In insect SF-21 cells, Drosophila IAP 1 (DIAP1) can
block apoptosis induced by the Drosophila caspase Drosophila
melanogaster Interleukin-1b-Converting Enzyme, insect sf-caspase-1, and
mammalian caspase-3. In mammals, different IAPs execute various ways to
regulate the function of caspases, and multiple BIR domains, even in the same
IAP, use distinctly different functions to inhibit different caspases. In
Fas/caspase-8-induced apoptosis, IAPs do not bind caspase-8 but inhibit its
substrate caspase-3 to execute their anti-apoptotic roles. In contrast, IAPs
carry out their caspase-suppression roles in three ways in the mitochondrial
pathway of caspase activation: (1) competitively bind pro-caspase-9 and
therefore interfere with the formation of apoptosome between pro-caspase-9 and
apoptotic protease activating factor 1; (2) directly bind caspase-9; and (3)
directly bind caspase-3. Overexpression of IAP is induced by Bax and other
pro-apoptotic Bcl-2 family proteins, known for their ability to target
mitochondria and induce cytochrome c release. In the ER stress-induced
apoptosis pathway, IAPs inhibit caspase-2 and caspase-9 by their BIR domain
binding to suppress apoptosis [9].Recent studies showed that IAP can inhibit the activity of caspases
by binding of their conserved BIR domains to the active sites of caspases in
vitro and in vivo. IAPs inhibit caspases by promoting the
degradation of active caspases, or by sequestering the caspases away from their
substrates [8]. In insect SF-21 cells, Drosophila IAP 1 (DIAP1) can
block apoptosis induced by the Drosophila caspase Drosophila
melanogaster Interleukin-1b-Converting Enzyme, insect sf-caspase-1, and
mammalian caspase-3. In mammals, different IAPs execute various ways to
regulate the function of caspases, and multiple BIR domains, even in the same
IAP, use distinctly different functions to inhibit different caspases. In
Fas/caspase-8-induced apoptosis, IAPs do not bind caspase-8 but inhibit its
substrate caspase-3 to execute their anti-apoptotic roles. In contrast, IAPs
carry out their caspase-suppression roles in three ways in the mitochondrial
pathway of caspase activation: (1) competitively bind pro-caspase-9 and
therefore interfere with the formation of apoptosome between pro-caspase-9 and
apoptotic protease activating factor 1; (2) directly bind caspase-9; and (3)
directly bind caspase-3. Overexpression of IAP is induced by Bax and other
pro-apoptotic Bcl-2 family proteins, known for their ability to target
mitochondria and induce cytochrome c release. In the ER stress-induced
apoptosis pathway, IAPs inhibit caspase-2 and caspase-9 by their BIR domain
binding to suppress apoptosis [9].XIAP, the best characterized IAP so far, is generally recognized as
the most potent endogenous caspase inhibitor. XIAP has three BIR domains, BIR1,
BIR2, and BIR3, which have high affinity but unequal functions to caspases. The
BIR2 domain and the linker region between BIR1 and BIR2 specifically bind and
inhibit caspase-3 and caspase-7. The BIR2 domain lies across the active site of
caspase-3 and inhibits the activity of caspase-3 by blocking its
substrate-binding pocket. Overexpression of the BIR1-2 fragment plays key roles
in Fas/caspase-8-induced apoptosis. The caspase-9 inhibitory activity of IAP
requires both the BIR3 and RING domain in the mitochondrial pathway. Without
physically binding to the active site of caspase-9, the BIR3 domain forms as a
heterodimer with monomeric caspase-9, thereby preventing the dimerization and
activation of caspase-9. As well as the trapping of caspase-9 in a monomeric
form, the BIR3 domain can keep the active site of caspase-9 in an inactive
conformation. Surprisingly, the BIR2, BIR3, or RING domains show no caspase
inhibitory activity alone [10].An emerging area of study in apoptosis is the critical contribution
of the ER in both mitochondrial and ER-specific apoptosis pathways. Caspase-2,
which is localized to the ER, is the proximal mediator in the ER stress-induced
apoptosis pathway with caspase-9. They activate caspase-3 and caspase-7 and in
turn cleave the caspase substrates. Although some IAPs are capable of binding
to and inhibiting caspases 3, 7, and 9, only cellular IAP 2 (c-IAP2) directly
binds and inhibits caspase-2 by its BIR2 domain. This result provides a novel
mechanism in the early stages of ER stress-induced apoptosis [9]. IAP could block the convergence point of multiple caspase activation
pathways and thus inhibit apoptosis. Caspases 2, 3, 7, and 9 can be suppressed
by some kinds of IAPs, whereas other mammalian caspases (1, 6, 8, and 10) are
known to be resistant to the inhibition. Different members of the IAP family
vary more or less in regulatory mechanisms. Cellular IAP 1 (c-IAP1) and c-IAP2
can also bind caspases 3, 7, and 9, but not with as tight an affinity as XIAP.
Melanoma IAP (ML-IAP) and ILP-2 are significantly inferior to XIAP in caspase
inhibition, and their BIR domain is most homologous to the BIR3 domain.
Research on neuronal apoptosis inhibitory protein (NAIP) has shown its
anti-apoptotic functions both in vivo and in vitro. In addition
to its inhibitory activities on caspase-3 and caspase-7, like XIAP, NAIP can
also use its BIR3 domain to interact with caspase-9, surprisingly in an
ATP-dependent pathway [11]. Interestingly, some recent research seems to prove that only a few
IAP proteins, like DIAP1 in Drosophila and XIAP in mammal, possess the
ability to inhibit caspase [12]. Eckelman and Salvesen suggested that c-IAP1
and c-IAP2, which can also bind caspases 3, 7, and 9 but with not as tight an
affinity as XIAP, lost or never acquired the caspase inhibitory ability. It was
also shown that neither of the BIR domains (BIR2 and BIR3) in c-IAP1 or c-IAP2
can inhibit caspases because of critical substitutions in the regions targeting
caspase inhibition in XIAP. The BIR domains of c-IAPs can be converted to tight
binding caspase inhibitors by substituting these critical residues with XIAP
residues. Thus, c-IAP1 and c-IAP2 could only execute their binding function to
caspase, rather than caspase inhibition [13]. Some IAPs that have only one or two BIR domains are found in many
species, like survivin and the BIR repeat-containing ubiquitin conjugating
enzyme (BRUCE) in mammal, Schizosaccharomyces pombe IAP and Saccharomyces
cerevisiae IAP in yeast, Caenorhabditis elegans IAP 1 (CeIAP1) and
CeIAP2 in nematode, and deterin in fly, and these IAPs are relatively weak
caspase inhibitors or play no roles in binding or inhibiting caspases. In some
studies, they were even distinguished from the forenamed IAPs as BIR-domain-containing
proteins, because of the differences in both the functions and structural
features of their BIR domains. The mechanism of these IAPs functioning as
mitotic regulators will be discussed.
IAP and the ubiquitination
process
As well as binding with the BIR domains, IAP can also inhibit the
activity of caspase in the ubiquitination process. Ubiquitination is a
post-translational protein modification procedure that plays important roles in
apoptosis and signal transduction. By operating the processes of ubiquitin
activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin
protein ligase (E3), target proteins are attached by ubiquitins. They are in
turn recognized and degraded by some proteasomes. The C-terminus RING domain of
IAP has been identified as the essential motif for the activity of ubiquitin
ligase (E3) that is sufficient to cause ubiquitylation and subsequent
proteasome-mediated proteolysis [14]. The RING domain in mammalian IAPs, including c-IAP1, c-IAP2, XIAP,
ML-IAP, as well as Drosophila DIAP1 and DIAP2, possesses E3 ligase
activities. The giant BIR-containing protein BRUCE is a unique IAP family
member with dual roles. Its N-terminal BIR domain can mediate substrate
binding, whereas the C-terminal UBC domain provides E2 activities. BRUCE has an
unusual ubiquitin conjugation system in that it could combine in a single
polypeptide ubiquitin conjugating (E2) with ubiquitin ligase (E3) activity,
forming a chimeric E2/E3 ubiquitin ligase [15]. Survivin is degraded by ubiquitin-targeted
proteolysis at the end of mitosis [16]. Even more recently, XIAP has also been
reported to ubiquitylate caspase-9 by itself [17].If certain IAPs can negatively regulate their own activity,
autoubiquitination will work and degrade itself. This process can lower the
effect of IAP and allow the cell to undergo apoptosis. In c-IAP2, either
full-length protein or its RING domain alone could execute E3 ligase activity in
vitro to promote autoubiquitination, as well as monoubiquitylation of
caspase-3 and caspase-7. A recent study also indicated that the C-terminal RING
domain of c-IAP1 is required for binding to XIAP and promoting XIAP degradation
in several cells [18]. c-IAP1 can heterodimerize with XIAP through a RING-RING
interaction so as to regulate the endogenous XIAP abundance and reduce it in a
proteasome-dependent pathway. By doing so, these two mechanisms (ubiquitination
and autoubiquitination) seem to work in counteraction to keep a fine balance.
IAPs appear to enhance the degradation of themselves or their targets in an
unclear regulatory mechanism.
IAP and IAP antagonists
IAP, which is at the center of virtually all apoptotic pathways, is
also subject to strict regulation through feedback mechanisms. A number of important
inhibitory factors that could counteract the anti-apoptotic activity of IAP
have been discovered. They are several kinds of endogenous pro-apoptotic
proteins that function on almost every kind of IAP [1]. Recently,
neutralization between IAP and IAP antagonists becomes to be a researching
mania for the treatment of cancers.These endogenous IAP binding proteins were first identified in Drosophila,
named Reaper, Grim, Hid, and Sickle and were shown to bind and inhibit DIAP1.
They were defined as critical inhibitors of IAP activity. Later, mammalian
counterparts of the IAP antagonists were identified, named second mitochondrial
activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) [19]
and high-temperature-regulated A2/Omi (HtrA2/Omi) [20], that are
mitochondrial-derived proteins, as well as X-linked IAP-associated factor 1
(XAF1) [21], a nuclear protein. In recent studies, G1 to S phase transition
protein/ polypeptide chain release factor 3 (GSPT1/eRF3) was also found to
associate with the ER in mammal [22], and Nma111p, a nuclear protein, plays a
crucial role in yeast apoptosis like its mammalian counterpart HtrA2/Omi [23].
The functional execution of these proteins seems to require normal induction of
apoptosis. Structural studies have shown the physical interactions between
these IAP binding proteins and IAP. The IAP binding proteins in Drosophila
and mammal all have a highly conserved homologous sequence, named the
IAP-binding motif domain, at the N-terminus that can bind IAP BIR domains so as
to mediate their pro-apoptotic function, at least in part, by competing for
interaction with IAP, thus displacing the bound caspases that are then free to
amplify the caspase cascade continuously. The regulatory mechanism of Smac/DIABLO and HtrA2/Omi has been
extensively characterized. During the disruption of the mitochondria, more than
40 regulators or executors involved in mammalian apoptosis might be released
simultaneously, including cytochrome c, endonuclease G, Apoptosis-inducing factor
IAP, and Smac/DIABLO and HtrA2/Omi. On release, Smac/DIABLO and HtrA2/Omi are
cleaved to become their activated forms that exist as arc-shaped dimers and
pyramid-shaped homotrimers, respectively. The structural study of molecular
recognition between Smac and IAP shows that the IAP-binding motif domain in
active Smac, which is only the first four residues (56–59) of the sequence, binds
across the third b-strand of the BIR3 domain and competitively inhibits the BIR3
domain from binding caspase-9, which also has an overlapped binding site the
same as Smac. HtrA2 also inhibits the function of XIAP by directly binding the
BIR3 domain, but with less affinity than Smac. The full-length protein of Smac
and its NH2-terminal peptides can also bind the BIR2 domain of XIAP to disrupt
the association between BIR2 and caspase-3 with slightly weaker affinity. This
mechanism might be related more to steric hindrance than competitive binding
[24]. The isoform of Smac/DIABLO, named Smac3, which is generated by
alternative splicing of exon 4, can also interact with the second and third BIR
domains of XIAP, just as Smac/DIABLO. Strikingly, only Smac3 can accelerate
XIAP autoubiquitination and destruction. Smac3-accelerated XIAP ubiquitination
is contingent on the physical association of XIAP with Smac3 and an intact RING
domain of XIAP. Smac3-stimulated XIAP destabilization is partly attributed to
its ability to enhance XIAP ubiquitination [25].In TNF-mediated up-regulation of IAP gene expression, TNFa can increase
mRNA and protein levels of c-IAP1, c-IAP2, and XIAP, but not ML-IAP or survivin
in tumor cell lines. IAPs act synergistically with TNF family members to
promote survival of tumor cells [26]. Correspondingly, IAP antagonists can
induce the autoubiquitination activity and rapid proteasomal degradation of
IAPs, like cIAP-1 and cIAP-2. Depending on TNF signaling and de novo
protein biosynthesis, IAP antagonists can also induce NF-kB-stimulated
production of TNFa that kill cells in an autocrine fashion [27,28].XAF1 is another IAP inhibitor that binds and sequesters XIAP in the
nucleus. It is ubiquitously expressed in normal tissues, but is present at low
or undetectable levels in many different cancer cell lines. Although XAF1 might
play key roles in mediating the apoptosis of cancer cells, it is still unclear
whether the inhibition of XIAP in the nucleus simply separates the XIAP from
cytosolic caspases or whether there are additional effects from XIAP located in
the nucleus [21]. As well as the endogenous IAP binding proteins, such as Smac or
HtrA2, several additional potential IAP antagonists are reported in the
development of targeted therapies directly against IAP in cancer. Some members
of the IAP family, including c-IAP2 and survivin, can be regulated by survival
cytokines, including interleukin-3, interleukin-5, and granulocyte/macrophage
colony stimulating factor [29]. Mitochondrial proteins, including glutamate
dehydrogenase, Nipsnap 3 and 4, caseinolytic peptidase X leucine-rich
pentatricopeptide repeat motif-containing protein, and 3-hydroxyisobutyrate
dehydrogenase, are newly described to interact with XIAP, mainly by way of
BIR2. Through the interaction, they are able to antagonize XIAP inhibition of
caspase-3 in vitro [30]. There are a lot of antisense oligonucleotides and small molecule
chemical IAP inhibitors that have been generated to study the regulatory
function of IAP, especially in tumor cells and therapies. In one of the classic
therapeutic strategies, antisense oligonucleotides are used to decrease the
target IAP mRNA and subsequently decrease the protein available for both XIAP
and survivin. Recently, Wang et al discovered a phenylurea-based XIAP
antagonist that can block the interaction of downstream effector caspases with
XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent,
Bcl-2/Bax-independent mechanism [31].
Complicated interaction
between IAPs, IAP antagonists, and caspases
Above all, the accepted regulatory model is that IAP can suppress
cellular apoptosis through the inhibition of caspases, whereas, in contrast,
some IAP antagonists like Smac/DIABLO can directly bind and provide inhibitory
activity to IAPs, particularly XIAP. However, the in vitro affinity that
many IAPs possess for caspases is much lower when compared with XIAP, raising
the possibility that the method by which they mediate cellular protection might
involve mechanisms beyond direct caspase inhibition [12]. Recently, some interesting findings have emerged to suggest that
this scheme might not be as simple as originally thought. If the binding of
Smac/DIABLO to XIAP neutralizes the cytoprotective effects of XIAP, how could
binding of XIAP to Smac/DIABLO block the death-promoting function of
Smac/DIABLO? Which of these two opposite directions is more important [32]? Do cytoprotective IAPs inhibit apoptosis through the neutralization
of IAP antagonists rather than by directly inhibiting caspases? It is a very
interesting hypothesis and much research has reached this conclusion. Green et
al showed that the expression of Orgyia pseudotsugata multicapsid
nucleopolyhedrovirus IAP (op-IAP) in mammalian cells can block the
activation of caspase-3. But surprisingly, instead of inhibiting caspase-3
directly, op-IAP executes its protection by binding to Smac/DIABLO efficiently,
thereby preventing Smac/Diablo-mediated inhibition of endogenous cellular IAP
proteins (such as XIAP), which may then continue to directly inhibit caspases.
Op-IAP also has the ability to ubiquitinate pro-apoptotic cellular proteins
such as Hid using both the RING domain and BIR2, which might play an important
role in the anti-apoptotic process [33].There are more distinct conclusions on mammalian IAP that could efficiently
bind Smac/Diablo so as to provide protection in cells that express other
caspase-inhibiting IAP, such as XIAP. ML-IAP, which is inferior to XIAP in
caspase inhibition, has a very high affinity for Smac. It can bind mature Smac
to form an ML-IAP-Smac complex and disrupt the endogenous interaction between
XIAP and mature Smac [34]. Survivin can manifest its cytoprotection by
physically associating with XIAP and forming a survivin-XIAP complex,
increasing XIAP stability against ubiquitination/proteasomal destruction and
synergistic inhibition of caspase-9 activation in vivo and in vitro
[35]. Ceballos-Cancino et al found that survivin could regulate the
specific release of mitochondrial inter-membrane protein Smac/DIABLO during
apoptosis that is induced by etoposide. And survivin could also stabilize the
cytosolic levels of released Smac/DIABLO by associating with Smac/DIABLO to
delay its release [36].The RING domain-bearing IAP can also mediate the polyubiquitination
of Smac/DIABLO by ubiquitin ligase (E3) activity. For example, DIAP1 was
reported to cause ubiquitylation of the Drosophila IAP antagonists Grim,
Hid, and Reaper. XIAP and c-IAP1 were found to mediate ubiquitylation of
Smac/DIABLO in vitro [16]. BRUCE’s activity to ubiquitylate Smac depends
on the catalytically active UBC domain and the correctly folded BIR domain that
binds the substrate Smac [15]. In addition to merely competing for binding sites, certain IAP
antagonists can also destabilize IAP and cause IAP degradation by proteasomes.
Reaper and UBCD1 in Drosophila caused degradation of DIAP1 in a
RING-dependent manner, presumably by promoting DIAP1 autoubiquitination and
degradation [37]. In another report, Hid was found to stimulate DIAP1
polyubiquitination and degradation, whereas Reaper and Grim could down-regulate
DIAP1 through mechanisms that do not require DIAP1 function, such as a
ubiquitin-protein ligase [37]. In contrast, Smac/DIABLO does not promote the
ubiquitin ligase activity of XIAP in the same way as Drosophila IAP antagonists.
It mainly potentiates apoptosis by simultaneously antagonizing caspase-IAP
interactions and repressing IAP ubiquitin ligase activities [38].A recent study even reached an alternative conclusion, that
caspase-3 could attenuate the inhibition of caspase-9 mediated by XIAP. During
the mitochondrion-mediated pathway, the initiator caspase-9 can be activated
and in turn activate caspase-3 and caspase-7. The activated caspase-3 then
activates caspase-9 by cleaving caspase-9 and forms a positive feedback
amplification loop to accelerate apoptosis. The short peptide motif that
cleaved at Asp330 in caspase-9 permits
caspase-9 to interact with IAPs. This shows that cleavage by caspase-3 does not
activate caspase-9, but enhances apoptosis by alleviating XIAP inhibition of
this apical caspase at Asp315 by autolytic cleavage [39].
IAP in cell cycle and signal transduction
Despite the suppression of the caspase pathway, IAP has been
reported in a variety of cellular processes including the cell cycle and signal
transduction.IAPs with only one BIR domain mainly function as regulators of the
cell cycle, such as: survivin in mammal; Sacch. cerevisiae IAP
and Schiz. pombe IAP in yeast; CeIAP1 and CeIAP2 in C. elegans;
and deterin in Drosophila. Survivin is a fascinating member of the IAP
family, with its dual roles in mitosis and apoptosis. It is a relatively weak
caspase inhibitor but serves critical roles in mitotic regulation. Survivin is
expressed in most human tumors, but is rarely detected in fully differentiated
normal cells. With the localization to mitotic spindles, survivin is necessary
for the assembly of metaphase spindles and promotes the stabilization of
microtubule-chromatin interactions. The direct biochemical functional analysis
in Xenopus egg extracts also justified this conclusion. Removal or
inhibition of survivin could cause the disruption of spindles [40]. The roles
of survivin make it a good prognostic marker and an attractive target for
cancer therapy. Most interestingly, it has been shown that overexpressed
c-IAP1, which localizes exclusively to nuclei in cells, can also modulate the
cell cycle, possibly by interfering with the mitotic functions of survivin.
These findings could have important implications for cancers in which c-IAP1
overexpression occurs [41].Recently, several IAPs have been shown to be involved in many
signaling cascades. However, the mechanisms involved in the regulation of the
IAP genes are not fully understood. In the NF-kB pathway, where it is
potentially seminal for development of inflammation-associated tumors,
c-IAP2-mucosa-associated lymphoid tissue 1 fusion protein could constitutively
activate NF-kB so as to contribute to human cancer. However, c-IAP2 and
mucosa-associated lymphoid tissue 1 alone do not possess the same activation
capacity. In the mitogen-activated protein (MAP) kinase pathway in human
endothelial cells, TNFa could induce the expression of c-IAP1 and c-IAP2 at the
transcriptional level. Thus, MAP kinases could participate in the inhibition of
apoptosis by the induction of c-IAPs. It is a MAP kinase-dependent and NF-kB-independent
process [42]. In the extracellular signal-regulated kinase pathway in colon
cancer, XAF1 expression was up-regulated by inhibition of the extracellular
signal-regulated kinase 1/2 pathway through transcriptional regulation, so that
the expression of XIAP would, conversely, be down-regulated [43]. In the
phosphoinositide 3-kinase/Akt signal pathway, which is strongly activated by
insulin, survivin is significantly decreased by wild-type Fragile Histidine
Triad (Fhit). Then, overexpression of constitutively active Akt will inhibit
Fhit-induced apoptosis by the loss of endogenous Fhit expression [44–46].
Promising clinical applications of IAP
Apoptosis has been accepted as a fundamental component in the
pathogenesis of cancer. It is known that dysfunction of the apoptotic pathway
and deregulated cellular proliferation will ultimately lead to carcinogenesis
and tumor progression. Many different apoptosis regulators have been documented
in rendering tumor cells resistant to apoptosis both in vivo and in
vitro, especially the most potential IAP. When IAP family members are
overexpressed, cancer cells no longer proceed to apoptosis and become
increasingly resistant to standard chemo- and radiation therapies [1,2].Many studies have established a circumstantial association between
IAPs and cancer. Pathological overexpression of several IAP family members has
been detected in several classes of human cancers. For example, the overexpression
of IAPs, such as XIAP, c-IAP1, c-IAP2, NAIP, and survivin, has been detected in
prostate cancer [47] and breast cancer [48]. A preferential cytoplasmatic
localization of c-IAP1 was observed in pancreatic carcinogenesis [49]. c-IAP2
is also overexpressed in pancreatic ductal adenocarcinomas [49]. The elevated
expression of XIAP has been investigated in esophageal cancer tissues and cell
lines, and compared with normal tissues. It raises the possibility that high
levels of IAPs might confer an insensitivity to apoptosis induction by
caspase-3 activation and promote tumorigenesis by keeping mutated cells alive
[50]. In human pancreatic carcinogenesis, c-IAP1 expression is constantly
high in pancreatic intraepithelial neoplasia lesions, as well as in a subset of
primary and metastatic pancreatic ductal adenocarcinomas. c-IAP2 is also
overexpressed and detectable in low- and high-grade pancreatic intraepithelial
neoplasia lesions [49]. Zhu et al postulated that survivin plays an
important role in the onset of gastric carcinoma and that high survivin
expression is an early event of gastric carcinoma [51]. Kluger et al
reported that overexpression of XIAP is up-regulated by pretreatment with
phenoxodiol in metastatic melanoma [52]. As well as the central regulatory
mechanisms of IAP suppression through direct caspase and pro-caspase
inhibition, IAPs appear to regulate NF-kB family transcriptional
activators which have also been associated with malignancy. The cIAPs have been
found to function as regulators of NF-kB signaling. Through their ubiquitin E3 ligase
activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NF-kB-inducing
kinase, the central Ser/Thr kinase in the non-canonical NF-kB pathway [27].
Due to the unique pathological overexpression of IAP that has been
documented in cancer, a novel and promising strategy is suggested to develop
targeted therapies directly against IAP for the treatment of malignancy.
Compared to conventional cancer chemotherapies, the starting point for
developing these more selective and less harmful anticancer drugs is to use
various IAP inhibitors to suppress IAP activities. Several approaches have been
taken to target and eliminate IAP functions, in order to attempt to re-establish
sensitivity, reduce toxicity, and improve efficacy of cancer treatment.
Recently, some small molecules, like deguelin and D,L-sulforaphane, have been
shown to down-regulate IAPs to release their inhibitory activity over
pre-existing active caspases present in cancer cells [48,53]. These targeted
therapies could better control the primary cancer by decreasing its chance of
developing to secondary malignancies, and can also be used in tumor treatment
in combination with standard cancer chemotherapies.Another possible anti-IAP therapeutic strategy is a molecular method
named RNA interference (RNAi). The core of the RNAi strategy is to deplete IAP
expression. Recent evidence has suggested that XIAP is a key determinant in the
chemoresistance of cancer cells. The small interfering RNA (siRNA) is
constructed and transferred into cancer cells, in order to block the
overexpression of IAPs and other proteins, like S-phase kinase-associated
protein-2. These processes evaluate the effect of siRNA on cellular apoptosis
[54,55]. Treatment with XIAP siRNA in combination with paclitaxel, cisplatin,
fluorouracil, and etoposide could efficiently decrease XIAP expression and
induce cellular apoptosis [50,56,57]. Survivin knockdown by RNAi leads to
growth rate inhibition of myeloma cells related to apoptosis induction and deep
cell-cycle disruption, and makes myeloma cells sensitive to conventional
antimyeloma agents [58]. The vector-based short hairpin RNAs can effectively
reduce the overexpression of survivin and Ki67 in renal cancer. They induce
apoptosis and are used as a new agent in renal cancer gene therapy [59].Interestingly, there are a number of publications showing IAP
expression in normal tissues and cells. Although survivin is usually not
expressed, c-IAP1, c-IAP2, and XIAP have been found broadly expressed at the
mRNA level within normal cells [60]. In normal pancreatic tissues, c-IAP1
expression is constantly as high as that measured in pancreatic cancer cells in
the same conditions. But a preferential cytoplasmatic localization of c-IAP1
has been observed in tumor tissues. This suggests that c-IAP1 might contribute
to the regulation of the apoptotic process in the normal and the neoplastic
pancreas, depending on its subcellular localization [49]. In normal tissues,
IAPs could have some potential physiological roles, such as the regulation of
the immune system [61], the response to cell damage [2,61], and cell survival
and differentiation [62]. Further investigation is needed to help us understand
the detailed physiological roles of IAPs in normal tissues.In summary, research into the extensive characterization of the
interaction between IAPs, caspases, and IAP antagonists in apoptosis is so
important and indispensable that it will afford the necessary theoretical
support in anticancer therapy.
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