Inhibitor of apoptosis proteins (IAPs) as regulatory factors of hepatic apoptosis

Cellular Signalling 25 (2013) 1970–1980

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Review

Inhibitor of apoptosis proteins (IAPs) as regulatory factors of hepatic apoptosis Kewei Wang a,⁎, Bingliang Lin b a b

Departments of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA Department of Infectious Diseases, Third Affiliated Hospital of Sun Yat-sen University, China

a r t i c l e

i n f o

Article history: Received 3 May 2013 Received in revised form 13 May 2013 Accepted 4 June 2013 Available online 11 June 2013 Keywords: IAPs Caspase Hepatic apoptosis

a b s t r a c t IAPs are a group of regulatory proteins that are structurally related. Their conserved homologues have been identified in various organisms. In human, eight IAP members have been recognized based on baculoviral IAP repeat (BIR) domains. IAPs are key regulators of apoptosis, cytokinesis and signal transduction. The antiapoptotic property of IAPs depends on their professional role for caspases. IAPs are functionally non-equivalent and regulate effector caspases through distinct mechanisms. IAPs impede apoptotic process via membrane receptor-dependent (extrinsic) cascade and mitochondrial dependent (intrinsic) pathway. IAP-mediated apoptosis affects the progression of liver diseases. Therapeutic options of liver diseases may depend on the understanding toward mechanisms of the IAP-mediated apoptosis. © 2013 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Discovery of IAPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular structure and antiapoptotic mechanisms of IAPs . . . . . . . . . . . . . Regulation of IAP expression . . . . . . . . . . . . . . . . . . . . . . . . . . . Brief introduction about respective IAPs . . . . . . . . . . . . . . . . . . . . . . 4.1. cIAP1 and cIAP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Tissue distribution and regulation of cIAP1 and cIAP2 . . . . . . . . 4.1.2. Molecular structure and antiapoptotic mechanism of cIAP1 and cIAP2 4.1.3. Pathophysiological function of cIAP1 and cIAP2 . . . . . . . . . . . 4.2. XIAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Tissue distribution and regulation of XIAP . . . . . . . . . . . . . 4.2.2. Molecular structure and antiapoptotic mechanism of XIAP . . . . . . 4.2.3. Pathophysiological function of XIAP . . . . . . . . . . . . . . . . 4.3. Survivin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Tissue distribution and regulation of Survivin . . . . . . . . . . . . 4.3.2. Molecular structure and antiapoptotic mechanism of Survivin . . . . 4.3.3. Pathophysiological function of Survivin . . . . . . . . . . . . . . . 4.4. Livin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.1. Tissue distribution and regulation of Livin . . . . . . . . . . . . . 4.4.2. Molecular structure and antiapoptotic mechanism of Livin . . . . . . 4.4.3. Livin as a target for new anticancer strategies . . . . . . . . . . . . 4.5. Apollon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1. Tissue distribution and regulation of Apollon . . . . . . . . . . . . 4.5.2. Molecular structure and antiapoptotic mechanism of Apollon . . . .

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Abbreviations: IAPs, inhibitor of apoptosis proteins; BIR, Baculovirus IAP repeat; FLIPs, FLICE-inhibitory proteins; Smac/Diablo, Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI; TNF, tumor necrosis factor; HCC, hepatocellular carcinoma; RIP1, receptor-interacting protein 1; HSP90, heat shock protein 90; CDK4, cyclin dependent kinase 4; CD2, cluster of differentiation 2; Rb, retinoblastoma; ASO, Antisense oligonucleotides; NF-κB, nuclear factor kappalight-chain-enhancer of activated B cells; TRAF2, TNF receptor-associated factor 2; APAF-1, apoptotic protease activating factor 1. ⁎ Corresponding author at: One Illini Dr., Peoria, IL 61605, USA. Tel.: +1 309 680 8617. E-mail address: [email protected] (K. Wang). 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.06.003

K. Wang, B. Lin / Cellular Signalling 25 (2013) 1970–1980

4.5.3. Pathophysiological function of Apollon . . . . . . . NAIP . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6.1. Tissue distribution and regulation of NAIP . . . . . 4.6.2. Molecular structure and antiapoptotic mechanism of 4.6.3. Pathophysiological role of NAIP . . . . . . . . . . 4.7. ILP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Clinical significance of IAPs and their relationship with liver diseases 6. Future study and summary . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6.

1. Introduction 1.1. Discovery of IAPs IAPs belong to a branch of antiapoptotic family. Prototype of IAPs was initially described as inhibitors of apoptosis in baculovirus with many homologues across species [1]. IAP family is highly conserved in yeast, nematodes, flies, invertebrates and vertebrates [2]. In human, eight IAP members have been ascertained according to baculoviral IAP repeat (BIR) domains consisted of 70 amino acid motifs in all of IAPs [3]. Neuronal apoptosis inhibitory protein (NAIP) is the first IAP member, which was thus called BIRC1 [4]. NAIP was found to be partly deleted in patients with the neurodegenerative disorder such as spinal muscular atrophy. NAIP blocks apoptotic cell death in response

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to treatment with menadione (a potent inducer of free radicals) or TNFα & cycloheximide intervention [4]. Other human IAPs were then identified by familial DNA databank searches and subsequently testified by their antiapoptotic capacity, including cIAP1/HIAP2/hMIHB/ BIRC2, cIAP2/HIAP1/hMIHC/BIRC3, XIAP/hILP/MIHA/BIRC4, Survivin/ BIRC5, Apollon/BRUCE/BIRC6, Livin/ML-IAP/BIRC7, and IAP-like protein 2/BIRC8, respectively (Fig. 1). The degree of apoptosis is tightly controlled by levels of endogenous IAPs in mammalian cells. IAPs exert their antiapoptotic function via directly protein binding of caspases as well as neutralization of Smac/DIABLO, which further activates downstream antiapoptotic cascades [5,6]. The apoptosis controls embryonic development, cell proliferation and tissue regeneration. Dysfunction or dysregulation of IAPs results in congenital anomalies and pathological conditions such as tumorigenesis, autoimmune diseases, neurodegenerative disorders and so on [7]. 2. Molecular structure and antiapoptotic mechanisms of IAPs

Fig. 1. IAPs belong to a branch of antiapoptotic family. Prototype of IAPs was initially described as inhibitors of apoptosis in baculovirus with many homologues across species. In human, eight IAP members have been ascertained according to baculoviral IAP repeat (BIR) domains consisted of 70 amino acid motifs in all of IAPs.

IAPs are defined by the presence of one or more repeats of a highly conserved BIR motif (Table 1). BIR protein domain is essential for the regulation of apoptosis, cytokinesis and signal transduction [8]. Although the family-defining BIR domain is highly conserved, distinct BIR domains, even within the same protein, have different functions. In addition to BIR domains, XIAP, cIAP1, cIAP2, Livin/ML-IAP and IAP-like protein 2 have a conserved RING domain at their C-terminal end that allows these proteins to act as E3 ubiquitin ligases [9]. The activity of E3 ubiquitin ligase in IAPs is capable of promoting ubiquitination and proteasomal degradation of caspases, TRAF2, and several other partners [10]. cIAP1 and cIAP2 contain a caspase-recruitment domain to mediate protein–protein interactions [11]. Some unique protein domains include a nucleotide-binding and oligomerization domain and a leucine-rich repeat in NAIP, nuclear export signal and coiled coil in Survivin, and an ubiquitin-conjugating domain in Apollon. Structure of IAPs is closely associated with their antiapoptotic function. The antiapoptotic property of IAPs depends on interaction between the BIR domains and caspases. XIAP, cIAP1 and cIAP2 can directly bind special areas of caspases-3, -7, and -9 [12–14]. For instance, the BIR3 domain of XIAP directly binds to the small subunit of caspase-9, but the BIR2 domain interacts with the active-site substrate binding pocket of caspases-3

Table 1 General characteristics of IAPs. Name

Domain

Disease

BIRC1, BIRC2, BIRC3, BIRC4,

NAIP cIAP1, AP11 cIAP2, AP12 XIAP, AP13

BIR-BIR-BIR-NOD-LRR BIR-BIR-BIR-UBA-CARD-Ring BIR-BIR-BIR-UBA-CARD-Ring BIR-BIR-BIR-UBA-Ring

BIRC5, BIRC6, BIRC7, BIRC8,

Survivin, AP14 Apollon, BRUCE Livin/ML-IAP, ML-IAP IAP-like protein 2

BIR-NES-CC BIR-UBC BIR-Ring BIR-UBA-Ring

Spinal muscular atrophy, type 1; Legionnaires' disease Multiple cancers; Liver disease MALT lymphoma; Liver disease X-linked lymphoproliferative disorder; Inflammatory bowel disease; Multiple cancers Multiple cancers MALT lymphoma Multiple cancers, especially melanoma

Note: BIR, baculovirus IAP repeat; NOD, nucleotide-binding and oligomerization domain; LRR, leucine-rich repeat; UBA, ubiquitin associated. CARD, caspase-recruitment domain; Ring, really interesting new gene; CC, coiled coil; NES, nuclear export signal; UBC, ubiquitin-conjugating.

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K. Wang, B. Lin / Cellular Signalling 25 (2013) 1970–1980

and -7 [15]. These bindings furthermore stimulate downstream signaling pathway such as Akt survival cascade and/or NF-κB activation, which can counteract apoptosis. Direct binding of caspase cubic structure by IAPs is an importantly regulatory means of caspases. The RING domain of IAPs targets other proteins such as caspase-3 and-7 for ubiquitination and degradation [16]. In mitochondria, Smac/Diablo released from the mitochondrial intermembrane space can antagonize the inhibitory effect of IAPs on caspases when Smac/Diablo binds to IAPs (e.g. XIAP) [17]. Smac/Diablo thus is a negative regulator of IAPs and unfolds its apoptosis-opposing property. Cytoprotective IAPs exert their effects through direct caspase binding, whereas the role of IAPs is weakened by neutralization of Smac/DIABLO. Antiapoptotic IAPs involve both membrane receptor-dependent cascade and mitochondrial dependent pathway (Fig. 2). 3. Regulation of IAP expression IAPs, as a group of signaling molecules, function in a wide range of cellular activities such as the inhibition of caspases and the promotion of cell-cycle progression. These diverse roles may be associated with many different domains that are well embedded within different proteins in the family. All of IAP family proteins have a common feature or suppressing cell death, but their expression in vivo appears to be regulated differentially (Table 2), thus presumably providing mechanisms for controlling the presence or absence of IAPs in response to particular cellular or environmental signals, e.g. levels of reactive oxygen species, concentration of intracellular Ca2+ [18,19]. In addition, a mechanism by which nuclear factors regulate expression of IAPs modulates hepatic apoptosis through transcriptional control, i.e. Foxa2/cIAP1 signaling pathway. Foxa2 can thus be a potential target for therapeutic intervention in

Table 2 Regulation of IAP expression. Target

Mechanism

Transcriptional nuclear factors

NF-κB/cIAP1, cIAP2, XIAP, and Survivin Foxa2/cIAP1; p53/Survivin; p53/Apollon; NF-κB/NAIP? IAP siRNA, e.g. XIAP/AEG35156 and Survivin/LY2181308 Smac/Diablo: AT-406; HtrA2/Omi: UCF-101 Reactive oxygen species, concentration of intracellular Ca2+

Post-translational/translational Mitochondrial pathway Cellular or inner environmental signals e.g. paracrine or autocrine

liver diseases [20]. Survivin signaling is mediated through NF-κB pathway during glycochenodeoxycholate-induced hepatocyte apoptosis [21]. Moreover, IAPs are post-transcriptionally modified by small interfering RNAs. The down-regulation of IAPs by siRNAs inhibited the expression of cartilage oligomeric matrix protein (COMP) that elevated the cell survival rate. Levels of mRNAs for most of the IAP family members were not increased by COMP, indicating that a translational/post-translational mechanism was involved in their induction [22]. IAP proteins regulate caspases to modulate apoptotic process. In turn, IAPs themselves can be neutralized by Smac/DIABLO, which, in response to apoptotic stimuli, is released from the mitochondria into the cytosol where it binds to IAPs. The combination of IAP-Smac/ DIABLO displaces caspases and consequently shifts the apoptotic signal direction [23,24]. For example, mitochondrial Smac/Diablo can bind to XIAP, which displaces caspases from XIAP and enables their activation. Cytoprotective IAPs, such as XIAP, ML-IAP and ILP-2, exert their antiapoptotic effects via both caspase binding and the neutralization of

Fig. 2. Molecular mechanisms of apoptosis. Hepatic apoptosis can be triggered by different causative factors such as such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response. Diverse stimuli from outside the cell may continually induce the activation of caspase-8, caspase-3, and DNA fragmentation through membrane receptors. The membrane receptor-mediated apoptosis is called the extrinsic pathway. Intracellular metabolic disturbances can cause mitochondrial damage and result in cytochrome c release and caspase-9 activation. The activated caspase-9 further stimulates the activation of caspase-3 and eventual apoptosis. The mitochondrion-mediated apoptosis is also named as mitochondrial pathway or intrinsic pathway. The mitochondrial pathway is different from the extrinsic apoptotic pathway, but two pathways are coexisted in liver. The mitochondrial pathway is often required to amplify the relatively weak death receptor-mediated apoptotic signals in liver.

K. Wang, B. Lin / Cellular Signalling 25 (2013) 1970–1980

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4. Brief introduction about respective IAPs

Table 3 Clinical trials of IAP antagonists. Name

Target

Disease

Status

Trial code

YM 155

Survivin

LY2181308

Survivin

AEG35156

XIAP

AT-406

XIAP, cIAP1, cIAP2

Prostate cancer Melanoma Lung cancer Breast cancer Non-Hodgkin's lymphoma Non-small cell lung cancer Prostate cancer Myeloid leukemia Refractory/relapsed AML Hepatocellular carcinoma Solid tumors Advanced solid tumors

Phase I/II Phase II Phase II Phase II Phase II Phase II Phase II Phase II Phase I/II Phase I/II Phase I Phase I

NCT00514267 NCT01009775 NCT00328588 NCT01038804 NCT01007292 NCT01107444 NCT00642018 NCT00620321 NCT00363974 NCT00882869 NCT00372736 NCT01078649

Smac/DIABLO. A delicate equilibrium between IAPs and Smac/DIABLO can result in a dynamic regulation of the apoptotic threshold [17,25]. Essential for the ability of Smac/Diablo to bind to IAPs and to release caspases is a conserved tetrapeptide motif which is also present in HtrA2/Omi, another mitochondrial proapoptotic factor [26,27], as well as in Drosophila proapoptotic proteins Reaper, Hid, Grim, and Sickle that can bind to Drosophila DIAP1 [28]. Regulation of IAP expression is finely balanced. The interaction between the BIR domains and caspases confers the antiapoptotic activity of IAPs. Direct inhibition of caspase activity by IAPs is certainly a very skillful means of regulation, when considered that signaling cascades mediated by proteolytic caspases is irreversible once activated and therefore must be precisely regulated in order to prevent inappropriate demise of cells.

Overexpression of IAPs has been shown to suppress apoptosis induced by a variety of stimuli, including TNFα, Fas, menadione, staurosporin, etoposide, Taxol, and growth factor withdrawal [4,29–31]. Enhanced IAPs, such as cIAP2 and XIAP by gonadotropin, can suppress apoptosis in granulosa cells, resulting in the development of follicles to the antral and preovulatory stages [31]. IAPs are increased in numerous malignancies. IAPs have been pursued as potential therapeutic targets for the treatment of cancers (Table 3). In past years, some small-molecule IAP antagonists have provided confirmative evidence for IAP biology and demonstrated their therapeutic potential. These inhibitory compounds act on multiple members of the family, such as XIAP, cIAP1 and cIAP2 simultaneously. Obvious proof not only has clarified the role of IAP proteins for apoptosis, but also has been instrumental in understanding how IAPs function in complicate pathways (Fig. 3). Here we put the recently revealed signaling pathways of IAPs in the context of the delicately balanced network of cell death and activation. The following subsets outline the pathophysiologic role of IAP family members, respectively. 4.1. cIAP1 and cIAP2 4.1.1. Tissue distribution and regulation of cIAP1 and cIAP2 Human cIAP1 and cIAP2 genes reside within ~7 kb of each other on chromosome 11q22–23 [32]. Their high degree of similarity likely arises from gene duplication. Therefore, cIAP1 and cIAP2 are discussed in same section. Tissue distributions of cIAP1 and cIAP2 are reportedly similar, but the relative expression of cIAP1 is generally higher. Expression of cIAP1 and cIAP2 is the highest in the kidney, small intestine,

Fig. 3. Regulation of apoptosis-signaling pathways. The apoptotic cell death requires the interplay of a multitude of factors. These related factors are organized in a tight and efficient manner to mediate apoptotic signaling. The activation of apoptosis causes cell death instantaneously unless the cell death is inhibited by potent antiapoptotic signals. A lot of antiapoptotic factors have been identified. The members of IAP family can suppress caspases along extrinsic or intrinsic pathways. The activity of IAPs is further modulated by mitochondrial proteins. Bcl-2 family mainly regulates the intrinsic pathway in cytoplasm. Transcription factors such as NF-κB and p53 mediate apoptosis through up- or downregulation of apoptosis-related gene expression in nuclei.

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liver, lung, and lowest in the central nervous system [32]. cIAP1 and cIAP2 are amply expressed in hepatocytes [33]. Cells from cIAP1deficient mice show enhanced levels of cIAP2 protein in the absence of increased cIAP2 mRNA. Transient transfection studies reveal that cIAP1 regulates the expression of cIAP2 by a posttranscriptional and E3-dependent mechanism. Posttranscriptional downregulation of cIAP2 is affiliated by the ubiquitin protein ligase cIAP1 in vivo [34].

4.1.2. Molecular structure and antiapoptotic mechanism of cIAP1 and cIAP2 cIAP1 and cIAP2 each contain a caspase-recruitment domain, which is thought to mediate protein–protein interactions. cIAP1 and cIAP2 function by binding the distal cell death proteases such as caspase-3 and caspase-7 [12,13]. cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells [35]. cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein [36]. Otherwise, RIP1 binds caspase-8 and induces apoptosis. Upregulation of cIAP1 and cIAP2 thus facilitates cell survival. cIAP proteins are frequently overexpressed in cancer and their levels are implicated in contributing to tumorigenesis, chemoresistance, and poor patient-survival [37]. Although best known for their ability to regulate caspases and apoptosis, cIAPs also influence signalling pathways that lead to the activation of NF-κB pathway [38,39]. cIAP1 and cIAP2 modulate NF-κB activation and suppress TNFα-mediated apoptosis [40]. Either cIAP1 or cIAP2 is required for proper RIP1 polyubiquitination and NF-κB activation subsequent to TNFα treatment [41]. cIAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-κB activation and oncogenesis [42].

4.1.3. Pathophysiological function of cIAP1 and cIAP2 Antiapoptosis is a basic function of cIAP1 and cIAP2. In Drosophila, DIAP1 and DIAP2 genes are important cell death regulators, with overexpression-of-function mutants displaying profound defects in developmental and death-preventing activities [43]. Apoptosis in the ovary is thought to play an important role in ovulation. In granulosa cells from preantral and early antral follicles, extensive apoptosis is associated with reduced protein levels of cIAP2 and XIAP. Gonadotropin treatment increases cIAP2 and XIAP protein content and suppresses apoptosis in granulosa cells, resulting in the development of follicles to the antral and preovulatory stages [31]. cIAP2 is induced by inflammatory cytokines in an NF-κB-dependent manner and overexpression of cIAP2 inhibits apoptosis. Hepatocyte survival is dependent on NF-κB activation and cIAP2 contributes significantly to this protection [33]. cIAP1 not only promotes murine HCC in cooperation with Myc, an oncogene that drives proliferation, but also promotes apoptosis through both p53-dependent and -independent mechanisms [44]. Like other cell types, Myc sensitizes hepatoblasts to diverse proapoptotic signals, and dampening of such signals is linked to tumorigenesis in other contexts. Effects of cIAP1 appear more pronounced in vivo, suggesting that microenvironmental effects associated with tumor expansion may be critical for its prosurvival activities. Perhaps this explains the variable effects of cIAP1 observed in previous in vitro studies; alternatively, nonapoptotic cIAP1 activities may contribute to its oncogenic role. However, the antiapoptotic activity of cIAP1 would be unlikely to cooperate with prosurvival Akt or Ras pathways [45]. The identification of cIAP1 as an oncogene is interesting in light of the controversial role of IAPs in modulating apoptosis in mammalian cells [46]. A large body of work shows that mammalian IAPs can suppress apoptosis, although most studies observe relatively modest effects despite overexpression in transient assays, and mice with knockout of single cIAP genes do not display substantial apoptotic defects [34]. Similarly, cIAP expression has been associated with various cancer phenotypes, but no cIAPs have been decisively linked to tumorigenesis in vivo [47,48].

4.2. XIAP 4.2.1. Tissue distribution and regulation of XIAP XIAP is the best-characterized member of the mammalian IAP family. XIAP mRNA is ubiquitously observed in adult and fetal tissues examined except peripheral blood leukocytes [49,50]. However, analysis of XIAP expression at the single cell lineage is still needed to determine whether differentiation or cell lineage-specific expression occurs. The suppression of XIAP levels by RNA interference sensitizes Bcl-xL-overexpressing cells to death receptor-induced apoptosis. During mitochondrial apoptotic events, Smac/DIABLO and Omi/HTRA2 are released from the mitochondrial intermembrane space [51]. Two death-signaling proteins target XIAP (also cIAP1 and cIAP2) and prevent it from binding to caspases. Thus, Smac/Diablo and Omi/HTRA2 are negative regulators of XIAP. 4.2.2. Molecular structure and antiapoptotic mechanism of XIAP XIAP gene was initially discovered with a 273 base pair fragment that encodes a RING zinc-finger domain located on the X chromosome at Xq24–25. XIAP, a 57-kDa protein, consists of three BIR domains, a RING-finger domain conferring ubiquitin protein ligase (E3) activity, and an evolutionarily conserved ubiquitin-binding domain [4,29]. XIAP functions as suppressor of apoptosis through binding and thereby inactivating certain caspases. BIR domains of XIAP are sufficient for the inhibition of caspases. The BIR2 domain inhibits caspase-3 and -7, while BIR3 binds to and inhibits caspase-9 [14,52]. BIR domains are functionally distinct. Both BIR domains use a two-site binding mechanism for potent caspase inhibition. One of these sites is a conserved surface groove found in BIR2 and BIR3 of XIAP. BIR1 facilitates binding of XIAP to the TGFβ-activated kinase binding protein 1, thereby mediating activation of NF-κB [53]. Small molecule XIAP inhibitors that bind and inhibit the BIR 2 or BIR 3 domain enter the process of clinical development. The RING domain utilizes E3 ubiquitin ligase activity and enables IAPs to catalyze ubiquination of self, caspase-3, or caspase-7 by degradation via proteasome activity [54]. XIAP stands out of the mammalian IAP protein family, because it is the only member capable of blocking active caspases. The inhibitory constants for XIAP, cIAP1, and cIAP2 were measured against caspases-3 and -7 range from 0.2 to 10 nm, indicating that the XIAP is the most potent human IAP protein currently identified [15,55]. The binding affinity of XIAP to caspases (-3 and -7) is very strong, whereas binding to the initiator caspase-9 is of considerably lower affinity and binding to the initiator caspase-8 is undetectable [14,56]. XIAP is modulated by up- or down-stream regulatory signals. XIAP inhibits caspase activation that requires mitochondrial amplification of death receptor signals. By acting upstream of mitochondrial activation, XIAP supports the long-term proliferative capacity of cells following CD95 stimulation. 4.2.3. Pathophysiological function of XIAP Apoptotic regulation is an extremely important biological function, as evidenced by the conservation of IAPs from Drosophila to humans. In granulosa cells from preantral and early antral follicles, extensive apoptosis was associated with reduced protein levels of XIAP and cIAP2 [31]. Gonadotropin treatment increased XIAP and cIAP2 protein content results in the development of follicles to the antral and preovulatory stages. Deregulation of XIAP can result in cancer, neurodegenerative disorders, and autoimmunity [57,58]. Currently, XIAP is the only cellular protein that potently inhibit the enzymatic activity of mammalian caspases at both the initiation phase (caspase-9) and the execution phase (caspase-3 and -7) of apoptosis. Given its role in apoptosis and its frequently elevated expression in malignant cells, XIAP has garnered the most attention as a promising therapeutic target in cancer. High proportions of XIAP may function as a tumor marker [59,60]. In the development of lung cancer NCI-H460, the overexpression of XIAP not only inhibits caspase, but also stops the activity of cytochrome c [61]. In developing prostate cancer, XIAP is one of four IAPs overexpressed in the prostatic epithelium [62]. HCC has similar unfavorable feature

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to other tumor types with an imbalance between unrestrained cell proliferation and impaired apoptosis. The mRNA ratio XIAP/XAF1 was significantly higher in HCC than in cirrhotic tissues. Moreover, high XIAP/ XAF1 ratio was an indicator of poor prognosis when overall survival was estimated and elevated XIAP immunoreactivity was significantly associated with shorter survival [63]. Small-molecule IAP antagonists have been synthesized as potential therapeutics for the treatment of cancers. siRNAs against XIAP and Survivin effectively reduce the level of XIAP and Survivin and increase therapeutic sensitivity to some chemotherapeutic agents. These siRNAs of both XIAP and Survivin had been used in clinical trial for human HCC [64]. Moreover, XIAP has important roles in a diverse set of nonapoptotic signaling pathways, such as NF-κB, MAP kinase, and the ubiquitin proteosome pathways, and in modulating a variety of cellular functions including immune regulation, cell division and differentiation, cell migration, morphogenesis, and heavy metal metabolism as described in other review [65]. Alterations in expression of XIAP result in cellular malfunctions in response to several intrinsic and environmental cues, which can also contribute to disease progression. In particular, XIAP seems to be a potent regulator of lymphocyte homeostasis. Defects in the XIAP gene can cause an X-linked lymphoproliferative disease [66]. Mutations in the XIAP gene can result in a severe and rare type of inflammatory bowel disease [67]. 4.3. Survivin 4.3.1. Tissue distribution and regulation of Survivin Survivin is expressed highly in fetal tissue and most tumors. In mouse embryo, prominent distribution of Survivin is found at embryonic day (E) 11.5, whereas at E15–E21, Survivin expression is restricted to only a few locations [30,68]. Expression of Survivin contributes to tissue homeostasis and differentiation in embryonic and fetal development. Survivin exhibits the most restricted expression of an IAP family member in adult tissues. Molecular mechanisms of Survivin regulation are still not well understood, but regulation of Survivin seems to be linked to the nuclear factors. NF-κB/Survivin signaling pathway modulates GCDC-induced hepatocyte apoptosis. Human Survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway [69]. Survivin interacts with HSP90, CDK4, CD2, and Rb/E2F complex as well [70]. Survivin is localized to the mitotic spindle by interaction with tubulin during mitosis and may play a contributing role in regulating mitosis. Survivin expressed in the G2-M phase is highly regulated by the cell cycle [71]. 4.3.2. Molecular structure and antiapoptotic mechanism of Survivin Survivin, the smallest mammalian IAP, is structural unique member of IAP family, containing a single BIR and a long carboxyl-terminus α-helix [72]. Survivin does not directly bind caspases. Instead, Survivin inhibits apoptosis via cooperative interactions with other partners to form IAP–IAP complexes in vivo [73]. Biochemical data reveal that Survivin BIR residues 15–38 are associated with discontinuous sites in XIAP BIR1 and BIR3 [73]. Only a pool of Survivin compartmentalized in mitochondria, and released in the cytosol in response to cell death stimuli, has the ability to associate with XIAP and this recognition is inhibited by Survivin phosphorylation on Ser20 by protein kinase A [74]. The Survivin–XIAP complex functionally enhances XIAP stability against ubiquitin-dependent degradation, synergistically increases the activity of XIAP for caspase inhibition, and directly participates in XIAP-mediated intracellular NF-κB activation [75]. The Survivin–XIAP complex may reciprocally govern Survivin stability through inhibiting RING-mediated polyubiquitination and proteasomal destruction of Survivin. Survivin also interacts with other large IAPs such as BRUCE and cIAP1, in the control of cytokinesis and the mitotic spindle checkpoint [73,76]. IAP–IAP complexes provide a general mechanism to expand the functional repertoire of these molecules. Other mechanisms of Survivin cytoprotection include the ability of mitochondria-localized Survivin to sequester pro-apoptotic Smac/

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DIABLO away from XIAP, or altogether prevent its release from mitochondria [77]. Therefore, Survivin cytoprotection involves a pathway of cytoplasmic-mitochondrial shuttling. 4.3.3. Pathophysiological function of Survivin The Survivin protein leads to negative regulation of apoptosis. Disruption of Survivin pathway results in increase in apoptosis and decrease in tumor growth [78]. Survivin is an ideal target for cancer therapy since cancer cells are targeted while normal cells are left alone. An attention is growing due to its universal over-expression in human tumors, its prominent role in disparate networks of cellular division, and intracellular signaling and antiapoptosis. Several preclinical studies have demonstrated that targeting Survivin expression by the use of small interfering RNAs, dominant negative mutants, antisense-oligonucleotides and small molecule repressors could sensitize tumor cells towards chemotherapy, irradiation, and suppression of tumor growth. Recent studies further revealed that radio-sensitization achieved by Survivin inhibition seems to be multifaceted and involves caspase-dependent and caspase-independent mechanisms [79]. Alterations in the expression of Survivin and Livin/ML-IAP are frequent in HCC cells [63]. Moreover, apoptosis is an important contributing factor during neuronal death in a variety of neurodegenerative disorders, including multiple sclerosis, Parkinson's disease and sciatic nerve injury. The Survivin serves a dual role in the inhibition of apoptosis as well as a vital role in mitosis and cell division. Due to the various roles of Survivin during cell division and apoptosis, targeting this protein illustrates a new therapeutic window for the treatment of neurodegenerative diseases [80,81]. Survivin is an immunotherapeutic target for adult and pediatric malignant brain tumors. 4.4. Livin 4.4.1. Tissue distribution and regulation of Livin Livin (ML-IAP, KIAP) is expressed in placenta, testes, spinal cord and lymph node under physiological conditions [82]. It is hard to be detected in most normal differentiated tissues, but is present in transformed cells and in several cancers such as carcinomas of the breast, cervix and prostate, melanoma tissues, and lymphoma cells [83]. Livin is also overexpressed in gastric cancer, colon cancer, pancreatic cancer, and hepatoma cells [84]. Livin is upregulated by viral oncogene E7 and also bound to E7 oncoprotein [85]. Manipulation of Livin expression may lead to the development of new immunotherapy and/or gene therapy strategies for the treatment of cancer. 4.4.2. Molecular structure and antiapoptotic mechanism of Livin Human Livin gene, located on chromosome 20 at band q13, comprises five introns and six exons within 46 kb region and produces a protein of 280 amino acids. Livin harbors a single BIR domain and a COOH-terminal RING finger domain. The respective three-dimensional structures of Livin-BIR and XIAP-BIR3 are very similar, including the peptide-binding sites of the structures [86]. The BIR domain forms a novel zinc-fold that is critical motif for antiapoptotic activity. The importance of RING domain is to promote IAP ubiquitylation and degradation by the proteasome. Human Livin has two alternatively spliced transcripts and can give rise to two different isoforms Livin-α and Livin-β [87]. The Livin-α protects cells from apoptosis induced by staurosporine. In contrast, apoptosis initiated by etoposide was blocked only by the β-isoform. The targeted inhibition of Livin-β blocks the growth of HeLa cells and the silencing Livin-β sensitizes those cells to different pro-apoptotic stimuli such as UV irradiation, TNFα, or etoposide [88]. In tumoral bladder tissues, only Livin-α is expressed in a proportion of tumors with a high risk of relapse [89]. Two isoforms strongly differ in their functional significance for the antiapoptotic ability in tumor cells and may represent a regulatory balance between apoptosis and its counterpart. These differences in biological activities may indicate the presence of critical amino acids outside the BIR and

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RING domains in Livin molecular structure. The mechanism of Livin antiapoptosis is controversial. The antiapoptotic activity of Livin may be attributed to its lone BIR domain [82]. Livin directly interacts with caspase-3 and -7 in vitro and caspase-9 in vivo via its BIR domain. The mutations in the BIR can reduce both inhibition of caspase-9 and its antiapoptotic activity. Nevertheless, the inhibitory effect of Livin on caspase-3 and caspase-9 is much weaker than that of XIAP. The antiapoptotic effect of endogenous Livin may be due to its antagonizing the XIAP–Smac interaction, rather than direct inhibition of caspase-9. Livin is negatively regulated by Smac/DIABLO. Livin exhibits E3 ubiquitin ligase activity to degrade the pivotal apoptotic regulator Smac/DIABLO through the ubiquitin–proteasome pathway [82,90]. A high-affinity between Livin and Smac may be associated aspartate 138 of Livin with alanine at the amino terminus in Smac. This affinity interaction can be eliminated by amino acid mutations in the BIR domain of Livin. Livin interaction with Smac can effectively compete with the XIAP–Smac interaction. Thus, Livin disrupts XIAP–caspase interaction and contributes to caspase inhibition, rather than a direct suppressor of caspases. 4.4.3. Livin as a target for new anticancer strategies Livin is preferentially expressed in tumor but almost not in normal adult tissues. Livin may serve as a valuable diagnostic marker to identify cancers. Level of Livin expression is associated with an aggressive phenotype. Livin expression can be sign of tumor progression and provides prognostically relevant information. High Livin expression in neoplasms is correlated with more aggressive behavior, such as decreased the response to chemotherapeutic agents and shortened survival time. Elevated expression of Livin renders melanoma cells resistant to drug-induced apoptosis [86]. Livin is an eligible target for immune-mediated tumor destruction. The peptide (KWFPSCQFLL) from the amino acid sequence of Livin can serve as a potent peptide vaccine for lung cancer [91]. High levels of the Livin protein may predict the progression of superficial bladder cancer and used as a marker of early recurrence. Livin plays a vital role in non-small-cell lung cancer development and the increased expression of Livin mRNA is similar to that of Survivin. Both Livin and Survivin together may be useful in inhibiting tumor growth through an increase in spontaneous apoptosis, and enhancing tumor cells response to apoptosis-inducing agents [92]. A comprehensive plan that combines chemotherapy, siRNA or ASOs, and vaccination will be developed in the treatment of various cancers. Livin and Survivin mRNAs are significantly overexpressed in HCC cancer tissues compared to non-neoplastic counterparts [63]. Knowledge about machinery of Livin regulation suggests that we may have strategies that ultimately inactivate this protein. Livin is a potential target for the development of drugs that selectively eliminate cancer cells. Methods to counteract Livin in tumor cells are practical application in the clinical treatment of certain cancer. Considerable efforts have been made to develop strategies for modulating apoptosis in cancer (leukemia, prostate and lung cancer) and apoptotic liver injury. 4.5. Apollon 4.5.1. Tissue distribution and regulation of Apollon BIRC6 (Apollon, BRUCE), initially identified in the mouse, is a giant membrane-associated IAP protein [93]. Tissue distribution of human Apollon is still under investigation now. Apollon is overexpressed in tumors such as brain gliomas, breast and ovarian cancers. BIRC6/ Apollon gene expression in childhood acute leukemia impacts therapeutic response and prognosis. BRUCE-knockout mice usually die perinatally due to impaired placental development that can be attributed to insufficient differentiation [76]. BIRC6 is highly conserved and its homologs have been identified in Drosophila (dBruce) and human (Apollon). Overexpression of dBruce can inhibit cell death induced by the proapoptotic Smac analogs Reaper and Grim. Depletion of Apollon causes defective abscission and cytokinesis-associated apoptosis,

accompanied by a block of vesicular targeting and defective formation of the midbody and the midbody ring. 4.5.2. Molecular structure and antiapoptotic mechanism of Apollon Apollon, a 528 kDa protein, is the largest member of the IAP family. Apollon contains one BIR domain at its N-terminal region and a unique UBC domain at C-terminus, which can form thioester bonds with ubiquitin [94]. Multifunctional Apollon depends upon the presence of different functional domains and multiple binding partners [95]. The single BIR domain at amino (N)-terminus of Apollon protein most closely resembles the BIR of Survivin. Apollon can counteract apoptosis by binding the active caspases via its single BIR domain [95]. However, its antiapoptotic function may be particularly relevant for the trans-Golgi network and vesicular structures where it mainly localizes [96]. It has also been suggested to function as an E3 ligase, and is itself regulated by ubiquitin-dependent degradation mediated by E2 UbcH5 and E3 Nrdp1 [97]. Near its carboxy (C)-terminal end, UBC domain endows the protein with a hybrid E2/E3 ubiquitin ligase activity [95]. Deletion of the C-terminal half of Apollon causes apoptosis in the placenta and yolk sac, leading to embryonic death. The mechanism is associated with upregulation of the tumor suppressor p53 and activation of mitochondrial apoptotic pathway that includes upregulated levels of Bax, Bak, and Pidd, translocation of Bax and caspase-2 onto mitochondria, release of cytochrome c and apoptosis-inducing factor [98]. Apollon facilitates the degradation of apoptotic proteins by ubiquitination. Apollon primarily mono- or oligo-ubiquitylates proteins, indicating that its main role is non-proteolytic [95]. Apollon exerts its cytoprotective activity by promoting ubiquitination and degradation of the pro-apoptotic protein Smac/DIABLO and by inhibiting caspase activity [97,99]. Apollon, unlike other IAPs, binds to the precursor of Smac and promotes its degradation. Apollon also binds to procaspase-9 and inhibits its cleavage. 4.5.3. Pathophysiological function of Apollon Apollon serves as mitotic regulator during cytokinesis. Ubiquitin is relocalized from midbody microtubules to the midbody ring and depletion of Apollon disrupts this process [76]. Apollon plays an important role in tumorigenesis. Apollon protein is overexpressed in brain cancers, e.g. gliomas. Brain cancer cell line SNB-78 expresses a high level of Apollon and shows resistance against various anticancer drugs [94]. Treating SNB-78 cells with antisense oligonucleotide against Apollon reduced the expression of Apollon protein and significantly sensitized the cells to apoptosis induced by cisplatin and camptothecin. Apollon protein is highly expressed in human ovarian cancer cells. An overexpression of Apollon is associated with poor prognosis in childhood acute myeloid leukaemia [100]. Apollon gene silencing induces apoptosis in breast cancer cells through p53 stabilization and caspase-3 activation. Apollon knockdown resulted in a marked, time-dependent decline of cell growth and an increased rate of apoptosis, which was associated with p53 stabilization and activation of the mitochondrial-dependent apoptotic pathway. Furthermore, the activation of caspase-3 seemed to be essential for the induction of apoptosis after Apollon knockdown. p53 stabilization and caspase-3 activation concur to determine the apoptotic response mediated by Apollon knockdown in breast cancer cells [101]. Antiapoptotic Apollon has been considered to be an attractive target with respect to molecular therapy of cancer, but targeting Apollon alone can be insufficient for the effective treatment of tumors overexpressing it. More effective therapies can be obtained as Apollon inhibitors are combined with anticancer drugs. The possibility of sensitizing tumor cells to apoptosis induced by some anticancer drugs through antisense oligonucleotide- or small interfering RNA (siRNA)-mediated down- regulation of Apollon has led to a new therapeutic target [101–103]. Apollon regulates apoptosis most likely through the mitochondrial pathway and are probably involved in multiple major human diseases, including cancers, where there is generally a failure of apoptotic mechanisms, and Parkinson's disease, where there is excessive death of critical neurons.

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4.6. NAIP

4.7. ILP-2

4.6.1. Tissue distribution and regulation of NAIP NAIP, known as BIRC1, is the first member of IAP family to be discovered. The expression of NAIP has been thoroughly investigated in the central nervous system, especially in the pattern of neurodegeneration such as spinal muscular atrophy [4,104]. NAIP also plays a role in non-neuronal cells. The presence of NAIP has been demonstrated in placenta, liver, spleen, lung, and peripheral blood leukocytes besides central nervous system [105]. NAIP is expressed at levels sufficient for detection by Northern blot analysis only in adult liver and in placenta, but can be detected in brain by RT–PCR [104]. Immunohistochemistry revealed that NAIP's presence in certain tissues, such as liver, lung, and spleen, which is most likely due to macrophage infiltration [106].

ILP2 (IAP-like protein 2) has previously been implicated in the control of apoptosis in the testis by direct inhibition of caspase-9. ILP2 is found only in the great apes. Functional residues of the Smac groove and caspase-9 interaction sites are completely conserved among human, gorilla and chimpanzee. On this basis it is likely that ILP2 is a functional protein. Therefore, to be an effective inhibitor in vivo, ILP2 would require a stabilizing protein or other influence to overcome its minimal structural integrity. Since expression of the protein is heavily restricted, such a stabilizer may be found only in testis. ILP2 contains BIR3 and RING domains, which is very similar to the C-terminal half of XIAP with 81% identity at the protein level. ILP2 is closely related to XIAP. Two auto-ubiquitination sites of XIAP, Lys322 and Lys328 in XIAP equivalent to Lys61 and Lys67 in ILP2, are maintained in ILP2 [119]. The crystal structure shows that all residues needed for caspase-9 and Smac interactions are conserved between ILP2 and XIAP. ILP2 can adopt an authentic BIR fold that contains an intact Smac binding pocket. However, the putative caspase-9 interaction domain is a surprisingly weak inhibitor and is also conformationally unstable. Comparison with the equivalent domain in XIAP demonstrated that the instability is due to the lack of a linker segment N-terminal to the inhibitory BIR domain. Over-expression of ILP2 had no protective effect on death mediated by Fas or tumor necrosis factor, but ILP2 potently inhibited apoptosis induced by over-expression of Bax or by co-expression of caspase-9 with APAF1, and pre- incubation of cytosolic extracts with ILP2 abrogated caspase activation in vitro [120]. A processed form of caspase-9 could be co-precipitated with ILP2 from cells, suggesting a physical interaction between ILP2 and caspase-9. Thus, ILP2 is an IAP family member with restricted specificity for caspase-9 [120]. Nevertheless, ILP2 is an unstable protein and cannot inhibit caspase-9 in a physiological way on its own. ILP2 may require assistance from unidentified cellular factors to be an effective inhibitor of apoptosis in vivo [25]. Because the native protein is minimally stable, the instability of the BIR domain may also mean that all potential BIR-dependent functions are lost in ILP2, including some of the signaling functions that have been proposed for other IAPs [121].

4.6.2. Molecular structure and antiapoptotic mechanism of NAIP NAIP contains at least four genes and repetitive elements which make it prone to rearrangements and deletions. The repetitiveness and complexity of the sequence make several tandem copies of NAIP sequences, most representing pseudogenes that vary in number among individuals [104]. NAIP protein with the molecular weight of 160 kDa has three BIR domains. Unique protein domains include a nucleotide-binding and oligomerization domain (NOD) and a leucine-rich repeat [107]. Pull down experiment showed direct interaction between the NOD domain of NAIP and the CARD-NOD domain of apoptotic protease activating factor 1 (APAF1) [108]. The integrity of the NOD domain is essential for effective inhibition of procaspase-9 and procaspase-3 cleavage by the NAIP protein. The apoptosis inhibitory ability of NAIP protein is attributed to the BIR domains supposedly through the inhibition of caspase-3 and -9 activities [108]. Alternatively spliced transcript variants encoding distinct isoforms have been found for NAIP gene [109]. The protein encoded by this gene contains regions of homology to two baculovirus inhibitor of apoptosis proteins, and it is able to suppress apoptosis induced by various signals. Full length wild type NAIP prevented the cleavage of both procaspase-9 to caspase-9 and procaspase-3 to caspase-3 in Hela cells. NAIP as an inhibitor of procaspase-9 is evolved to prevent apoptosis right at the initiation stage of apoptosome formation and this inhibition cannot be antagonized by Smac-type proteins [105]. 4.6.3. Pathophysiological role of NAIP NAIP is a modifier of spinal muscular atrophy (SMA) that is a common and lethal autosomal recessive neurodegenerative disorder caused by mutations in a neighboring gene SMN1 [104]. Consistent with its role as a modifier of SMA severity, NAIP has been shown to inhibit apoptosis by binding activated caspases [4]. Considering the co-expression of NAIP and XIAP in certain cells especially the neuronal cells, inhibition of casapse-9 activity by these proteins can be accumulative. The ultimate destiny of the cells with respect to apoptosis is dependent upon endogenous levels of these IAPs as compared to the activity of caspase-9 and Smac. In PC12 cells, caspase-9 activity was decreased upon differentiation due to down regulation of APAF-1. This allowed better regulation of apoptosis by both NAIP and XIAP, which are expressed in these cells [110]. Moreover, the effect of NAIP expression in other neurodegenerative diseases, such as Alzheimer's disease, Down syndrome, multiple sclerosis, and Parkinson's disease, has also been demonstrated [111–114]. The expression of NAIP was also significantly increased in clinical samples of prostate cancer from patients receiving androgen deprivation therapy. Increased expression of NAIP corresponded to increased DNA-binding activity of NF-κB. Expression of NAIP may be associated with enhanced survival of prostate cancer in response to castration [115,116]. In addition, polymorphism of a particular NAIP copy in mouse strains determined permissiveness of Legionella pneumophila replication in host macrophages [117]. NAIP-mediated L. pneumophila restriction is caspase-1-dependent, which results in the rapid death of infected cells [118].

5. Clinical significance of IAPs and their relationship with liver diseases IAPs have multiple physiological functions and clinical implications in some diseases. Antiapoptotic role of IAPs has been broadly investigated during carcinogenesis [8]. The disturbance of IAPs has been applying in several clinical trials (Table 3). Levels of IAPs can be used as diagnostic markers for cancer. Patients with hepatocellular carcinoma (HCC) have an increased expression of cIAP1, cIAP2, NAIP, XIAP and Survivin, as compared with the corresponding cirrhotic tissues [44,122]. Survivin was detected in all HCC. In samples positive for NAIP or Survivin, splice forms of these IAPs were almost always found as well [63]. Alterations in the expression of IAP family members, including Survivin and Livin/ ML-IAP, are frequent in HCC. Expression of IAPs is downregulated not only as therapeutic approaches for the treatment of cancer, but as prognostic indicator of cancer patients as well. A study demonstrated that Survivin and Livin/ML-IAP mRNAs were significantly overexpressed in HCC tissues. A significant relation was found between higher Survivin mRNA level and tumor stage, tumor grade and vascular invasion. The mRNA ratio XIAP/XAF1 was significantly higher in HCC than in cirrhotic tissues. Additionally, high XIAP/XAF1 ratio was an indicator of poor prognosis [63]. An imbalance in XIAP/XAF1 mRNA expression levels was correlated to overall patient survival and high XIAP immunoreactivity was a poor prognostic factor that was significantly associated with short survival. Chronic liver diseases are one of the most important death causes worldwide. 30,558 patients died from the chronic liver disease and cirrhosis, according to the 2009 USA National Vital Statistics Report. Chronic liver disease can be induced by various etiological factors such

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as alcohol, drugs, viruses, cholestasis, etc. Alcohol abuse and chronic ethanol consumption are major causes of liver fibrosis, accounting for more than 50% of all cirrhotic livers in Western countries. American Liver Foundation reported that nearly 20% of Americans have fatty liver, and of that number about 5% have nonalcoholic steatohepatitis. In children less than two years of age, end stage liver disease from biliary malformations account for over 75% of the liver transplantation. No matter the type of liver diseases, final outcomes are cirrhosis and liver failure. Liver transplantation is only way to cure liver cirrhosis and failure. Currently, near 16,000 liver-failure patients are on the waiting list for transplantation in the adult U.S. population. Because of the long waiting time for liver transplantation, 24% of the adult and 12% of the pediatric patients die from disease progression before transplantation. Apoptosis is a typical pathological feature in chronic liver diseases. Hepatic apoptosis and its deleterious effects are pivotal steps in the progression of most forms of liver injury. By examining the mechanisms by which components of the apoptotic machinery contribute to pathogenic processes, we will broaden an understanding with the liver repair response. Inhibition of the hepatic apoptosis can delay the progression of liver disease, reduce the morbidity of liver insufficiency, and prolong patient survival. For the candidates of liver transplantation, the interruption of hepatic apoptosis may extend their liver reserve function until a donor liver becomes available. Preliminary data had showed that upregulation of IAPs could alleviate liver injury and improved liver function [20,123–125]. However, clinical application of IAPs and their regulators is limited in chronic liver diseases, since the responsible mechanisms are still under investigation. Moreover, IAPs pitch in the pathogenesis and progression of several diseases such as neurodegenerative disorders, autoimmune diseases and others. IAPs are involved in growth regulation of activated lymphocytes. If this regulatory process is failed, the defect in homeostasis leads to autoimmunity and/or lymphoma. 6. Future study and summary Pathogenesis and progress of liver diseases are regulated by apoptosis. Therefore, pharmacological manipulation of the major apoptosis regulators, such as IAPs, TNF death receptor family, caspase inhibitors, antagonists of the p53–MDM2 interaction, NF-κB and PI3K pathways, and Bcl-2 family members, represent clinically attractive avenues towards effective therapeutic strategies [124,126]. Especially, transcriptional regulation of IAPs has been investigating in recent years. Roles of Foxa2, HNF6, C/EBPβ and NF-κB have been noticed in hepatic apoptosis [20,127,128]. Moreover, posttranscriptional modification by siRNA and/or chemical factors has been examined as well. These studies aim to discover potential targets through the interaction between nuclear factors and expression of IAPs. Hepatic apoptosis has quite different mechanisms due to distinct etiology such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response. Liver-protective mechanism of IAPs needs to be clarified in diverse functional models. The future study will, employed different models of hepatic apoptosis, investigate expressional profile of IAPs, transcriptional regulation of IAPs, and potential clinical use of IAPs. Resultant data can provide the scientific basis for further clinical trials that will evaluate the salvaging effects of IAPs on liver injury as well as examine the role of IAPs in liver regeneration and/or fibrosis. Long term goal is to investigate hepatoprotective effects of comprehensive role of IAPs and their regulatory mechanisms. This analysis will a) identify special target for therapeutic intervention in chronic liver diseases, b) extend hepatic functional reserve of cirrhotic patients until a donor liver becomes available, c) reduce the need for transplantation, and d) improve the patient quality of life. Ongoing study will expand the understanding for mechanisms of liver injury/ repair response, toward the goal of designing future hepatoprotective strategies for patients with liver failure. Specific strategies should be utilized to address the relationship between apoptosis and different liver diseases. For the treatment of premature cell death, the inhibition of proapoptotic key components such as

IAPs might be promising. Interventions in hepatic apoptosis can help delay disease progression and prolong patient survival. For tumorigenesis, strategies may include the targeted activation of proapoptotic tumor suppressors and/or alternatively the blockade of antiapoptotic oncogenes. From an understanding of the core components of the apoptosis mechanism at the molecular and structural levels, a lot of attempts to use IAPs as targets have been concentrated for anticancer therapy. Some new drugs targeting IAPs include: (i) Survivin inhibitor YM155, LY2181308, Survivin/Hsp90 antagonist shephedrin; (ii) XIAP-antisense oligonucleotide 1396–11, 1396–12, 1396–28, triptolide, AEG35156 and phenoxodiol targeting XIAP (a synthetic derivative of plant isoflavone genistein); (iii) AT-406 targeting cIAP1, cIAP2, and XIAP; (iv) cIAP1 antagonists OSU-03012 and ME-BS. The effectiveness of cancer therapy was the highest when several IAPs were down-regulated simultaneously, suggesting that multiple IAPs rather than an individual IAP member should be targeted. Potential problems with the long term clinical use of IAP inhibitors: (a) carcinogenesis; (b) upregulation of IAP independent cell events; (c) Biochemical flare or overshoot when stopped. These studies will clarify the detail of apoptotic/antiapoptotic signaling network. Possible methods include analysis of gene expression, novel proteomic approaches, as well as functional studies of theses apoptosis-related genes. Preferences are: 1) to amplify antiapoptotic role of IAPs during liver injury and repair; 2) combinative effects, such as saRNA of IAPs and caspase inhibitors for apoptotic liver injury; 3) to inhibit antiapoptotic role of multiple IAPs in cancer cells simultaneously. A comprehensive guideline should be considered, which will include: a) removal of causes (viral killers, alcohol abstinence, drug safety, diet); b) regulation of apoptosis by small non-coding RNAs (e.g. siRNA, saRNA) or caspase inhibitors; and c) replacement sick cells by activated hepatocytes or stem cells. In summary, apoptosis is an essential process in the pathogenesis of liver disease. The hepatic apoptosis and its regulatory mechanism can provide the necessary tools to combat liver diseases. Therapeutic strategies to promote organ regeneration and tissue repair after liver injury are able to improve the quality of life and prolong the survival of patients. References [1] M. Rothe, M.G. Pan, W.J. Henzel, T.M. Ayres, D.V. Goeddel, Cell 83 (7) (1995) 1243–1252 (Epub 1995/12/29). [2] A.G. Uren, M. Pakusch, C.J. Hawkins, K.L. Puls, D.L. Vaux, Proceedings of the National Academy of Sciences of the United States of America 93 (10) (1996) 4974–4978 (Epub 1996/05/14). [3] C. Sun, M. Cai, A.H. Gunasekera, R.P. Meadows, H. Wang, J. Chen, et al., Nature 401 (6755) (1999) 818–822 (Epub 1999/11/05). [4] P. Liston, N. Roy, K. Tamai, C. Lefebvre, S. Baird, G. Cherton-Horvat, et al., Nature 379 (6563) (1996) 349–353 (Epub 1996/01/25). [5] C. Du, M. Fang, Y. Li, L. Li, X. Wang, Cell 102 (1) (2000) 33–42 (Epub 2000/08/10). [6] C.J. Hawkins, J. Silke, A.M. Verhagen, R. Foster, P.G. Ekert, D.M. Ashley, Apoptosis: An International Journal on Programmed Cell Death 6 (5) (2001) 331–338 (Epub 2001/08/03). [7] K. Wang, B. Lin, ISRN Hepatology 2013 (2013) 14. [8] E.C. LaCasse, D.J. Mahoney, H.H. Cheung, S. Plenchette, S. Baird, R.G. Korneluk, Oncogene 27 (48) (2008) 6252–6275 (Epub 2008/10/22). [9] E.C. Dueber, A.J. Schoeffler, A. Lingel, J.M. Elliott, A.V. Fedorova, A.M. Giannetti, et al., Science 334 (6054) (2011) 376–380 (Epub 2011/10/25). [10] Y. Nakatani, T. Kleffmann, K. Linke, S.M. Condon, M.G. Hinds, C.L. Day, The Biochemical Journal 450 (3) (2013) 629–638 (Epub 2012/12/25). [11] H.H. Cheung, S. Plenchette, C.J. Kern, D.J. Mahoney, R.G. Korneluk, Molecular Biology of the Cell 19 (7) (2008) 2729–2740 (Epub 2008/04/25). [12] B.P. Eckelman, G.S. Salvesen, The Journal of Biological Chemistry 281 (6) (2006) 3254–3260 (Epub 2005/12/13). [13] N. Roy, Q.L. Deveraux, R. Takahashi, G.S. Salvesen, J.C. Reed, The EMBO Journal 16 (23) (1997) 6914–6925 (Epub 1998/01/31). [14] F. Cossu, M. Milani, P. Vachette, F. Malvezzi, S. Grassi, D. Lecis, et al., PloS One 7 (11) (2012) e49527 (Epub 2012/11/21). [15] R. Takahashi, Q. Deveraux, I. Tamm, K. Welsh, N. Assa-Munt, G.S. Salvesen, et al., The Journal of Biological Chemistry 273 (14) (1998) 7787–7790 (Epub 1998/05/09). [16] Y.L. Yang, X.M. Li, Cell Research 10 (3) (2000) 169–177 (Epub 2000/10/14). [17] D. Vucic, M.C. Franklin, H.J. Wallweber, K. Das, B.P. Eckelman, H. Shin, et al., The Biochemical Journal 385 (Pt 1) (2005) 11–20 (Epub 2004/10/16). [18] S.L. Mehta, N. Manhas, R. Raghubir, Brain Research Reviews 54 (1) (2007) 34–66 (Epub 2007/01/16). [19] E.A. Mercer, L. Korhonen, Y. Skoglosa, P.A. Olsson, J.P. Kukkonen, D. Lindholm, The EMBO Journal 19 (14) (2000) 3597–3607 (Epub 2000/07/19).

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