Novel Polypyridyl chelators deplete cellular zinc and destabilize the X-linked inhibitor of apoptosis protein (XIAP) prior to induction of apoptosis in human prostate and breast cancer cells

X-linked inhibitor of apoptosis protein (XIAP), inhibits the initiation and execution phases of the apoptotic pathway. XIAP is the most potent member of the inhibitor of apoptosis protein (IAP) family of the endogenous caspase inhibitors. Therefore, targeting XIAP may be a promising strategy for the treatment of apoptosis-resistant malignancies. In this study, we systematically studied the relationships of chemical structures of several novel ligands to their zinc (Zn)-binding ability, molecular target XIAP, and tumor cell death-inducing activity. We show that treatment of PC-3 prostate cancer and MDA-MB-231 breast cancer cells with these membrane-permeable Zn-chelators with different Zn affinities results in varying degrees of XIAP depletion. Following decreased level of XIAP expression, we also show apoptosis-related caspase activation and cellular morphological changes upon treatment with strong Zn-chelators N4Py and BnTPEN. Addition of Zn has a full protective effect on the cells treated with these chelators, while iron (Fe) addition has only partial protection that, however, can be further increased to a comparable level of protection as Zn by inhibition of ROS generation, indicating that cell death effects mediated by Fe- but not Zn-complexes involve redox cycling. These findings suggest that strong Zn-chelating agents may be useful in the treatment of apoptosis-resistant human cancers.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

Inhibitors of apoptosis proteins (IAPs) as potential molecular targets for therapy of hematological malignancies

Apoptosis, a programmed cell death, plays a key role in the regulation of tissue homeostasis. However, impairment of its regulation may promote formation and progression of malignancy. An important part of the apoptotic machinery are the inhibitor of apoptosis protein (IAP) family, regulating caspase activity, cell division or cell survival pathways through binding to their baculovirus AIP repeat (BIR) domains and/or by their ubiquitin-ligase RING zinc finger (RZF) activity. The following IAPs have been described so far: NAIP (neuronal apoptosis inhibitory protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome binding IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8). Several studies suggested a potential contribution of IAPs to oncogenesis and resistance to anti-tumor treatment. Increased IAP expression was found in variety of human cancers, including hematological malignancies, such as leukemias and B-cell lymphomas. A correlation between the progression of those diseases and high levels of survivin or XIAP has been reported. Overexpression of XIAP in acute myeloid leukemia or survivin in acute lymphoblastic leukemia and diffuse large B-cell lymphoma have been indicated as an unfavorable prognostic factors. Elevated cellular levels of cIAP1, cIAP2, XIAP and survivin correlated with a progressive course of chronic lymphocytic leukemia. Thus, targeting IAPs with small-molecule inhibitors by their antisense approaches or natural IAP antagonist mimetics, may be an attractive strategy of anti-cancer treatment. Such agents can either directly induce apoptosis of tumor cells or sensitize them to other cytotoxic agents, hence overcoming drug-resistance. This review demonstrates the current knowledge on IAP molecular biology, as well as the mechanisms of action and the development of IAP-targeting agents for treatment of hematological malignancies.     

Clioquinol induces cytoplasmic clearance of the X-linked inhibitor of apoptosis protein (XIAP): therapeutic indication for prostate cancer

Clioquinol (5-chloro-7-iodo-8-quinolinol) is a copper ionophore that was used primarily during the 1950-1970s as an oral antimicrobial agent. It has been established that clioquinol displays toxicity towards malignant cells, inducing caspase-dependent apoptosis. In the present study we therefore investigated the effect of clioquinol on the XIAP [X-linked IAP (inhibitor of apoptosis protein)], as one of its primary functions is to hinder caspase activity and suppress apoptotic cell death. Clioquinol treatment caused cytoplasmic XIAP to rapidly relocate to the nucleus in multiple human transformed (hyperplasic and carcinoma) prostate lines. Clioquinol also caused the cytoplasmic clearance of other IAP family members (cIAP1 and cIAP2). Copper, and no other relevant bivalent metal (e.g. zinc or iron), was exclusively required for clioquinol to elicit an effect on XIAP. We further demonstrated that clioquinol selectively targets and rapidly destroys transformed prostate lines without harming primary prostate epithelial cells. The toxicity of clioquinol was copper-dependent, positively correlated with the level of extracellular copper and could be abrogated by using the copper chelator TTM (tetrathiomolybdate). Clioquinol forced the profound accumulation of intracellular copper with ensuing toxicity influenced by key regulators of cellular copper homoeostasis. Taken together, our results provide significant insight into clioquinol toxicity and reveal an exciting therapeutic approach for the treatment of prostate cancer.     

Crystal structure of the BIR1 domain of XIAP in two crystal forms

X-linked inhibitor of apoptosis (XIAP) is a potent negative regulator of apoptosis. It also plays a role in BMP signaling, TGF-beta signaling, and copper homeostasis. Previous structural studies have shown that the baculoviral IAP repeat (BIR2 and BIR3) domains of XIAP interact with the IAP-binding-motifs (IBM) in several apoptosis proteins such as Smac and caspase-9 via the conserved IBM-binding groove. Here, we report the crystal structure in two crystal forms of the BIR1 domain of XIAP, which does not possess this IBM-binding groove and cannot interact with Smac or caspase-9. Instead, the BIR1 domain forms a conserved dimer through the region corresponding to the IBM-binding groove. Structural and sequence analyses suggest that this dimerization of BIR1 in XIAP may be conserved in other IAP family members such as cIAP1 and cIAP2 and may be important for the action of XIAP in TGF-beta and BMP signaling and the action of cIAP1 and cIAP2 in TNF receptor signaling.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.     

XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage

The IAP (inhibitor of apoptosis) family of anti-apoptotic proteins regulates programmed cell death. Of the six known human IAP-related proteins, XIAP is the most potent inhibitor. To study the mechanistic effects of XIAP on DNA damage-induced apoptosis, we prepared U-937 cells that stably overexpress XIAP. The results demonstrate that XIAP inhibits apoptosis induced by 1-[beta-d-arabinofuranosyl]cytosine (ara-C) and other genotoxic agents. XIAP had no detectable effect on ara-C-induced release of mitochondrial cytochrome c and attenuated cleavage of procaspase-9. In addition, we show that ara-C induces the association of XIAP with the cleaved fragments of caspase-9 and thereby inhibition of caspase-9 activity. The results also demonstrate that ara-C induces cleavage of procaspase-3 by a caspase-8-dependent mechanism and that XIAP inhibits caspase-3 activity. These results demonstrate that XIAP functions downstream of procaspase-9 cleavage as an inhibitor of both proteolytically processed caspase-9 and -3 in the cellular response to genotoxic stress.     

The LEF1/CYLD axis and cIAPs regulate RIP1 deubiquitination and trigger apoptosis in selenite-treated colorectal cancer cells

Inhibitor-of-apoptosis protein (IAP) inhibitors have been reported to synergistically reduce cell viability in combination with a variety of chemotherapeutic drugs via targeted cellular IAP (cIAP) depletion. Here, we found that cIAP silencing sensitised colorectal cancer (CRC) cells to selenite-induced apoptosis. Upon selenite treatment, the K63-linked ubiquitin chains on receptor-interacting protein 1 (RIP1) were removed, leading to the formation of the death-inducing complex and subsequent caspase-8 activation. Although the ubiquitinases cIAP1 and cIAP2 were significantly downregulated after a 24-h selenite treatment, cylindromatosis (CYLD) deubiquitinase protein levels were marginally upregulated. Chromatin immunoprecipitation assays revealed that lymphoid enhancer factor-1 (LEF1) dissociated from the CYLD promoter upon selenite treatment, thus abolishing suppression of CYLD gene expression. We corroborated these findings in a CRC xenograft animal model using immunohistochemistry. Collectively, our findings demonstrate that selenite caused CYLD upregulation via LEF1 and cIAP downregulation, both of which contribute to the degradation of ubiquitin chains on RIP1 and subsequent caspase-8 activation and apoptosis. Importantly, our results identify a LEF1-binding site in the CYLD promoter as a potential target for combinational therapy as an alternative to cIAPs.     

cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

Three members of the IAP family (X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins-1/-2 (cIAP1 and cIAP2)) are potent suppressors of apoptosis. Recent studies have shown that cIAP1 and cIAP2, unlike XIAP, are not direct caspase inhibitors, but block apoptosis by functioning as E3 ligases for effector caspases and receptor-interacting protein 1 (RIP1). cIAP-mediated polyubiquitination of RIP1 allows it to bind to the pro-survival kinase transforming growth factor-β-activated kinase 1 (TAK1) which prevents it from activating caspase-8-dependent death, a process reverted by the de-ubiquitinase CYLD. RIP1 is also a regulator of necrosis, a caspase-independent type of cell death. Here, we show that cells depleted of the IAPs by treatment with the IAP antagonist BV6 are greatly sensitized to tumor necrosis factor (TNF)-induced necrosis, but not to necrotic death induced by anti-Fas, poly(I:C) oxidative stress. Specific targeting of the IAPs by RNAi revealed that repression of cIAP1 is responsible for the sensitization. Similarly, lowering TAK1 levels or inhibiting its kinase activity sensitized cells to TNF-induced necrosis, whereas repressing CYLD had the opposite effect. We show that this sensitization to death is accompanied by enhanced RIP1 kinase activity, increased recruitment of RIP1 to Fas-associated via death domain and RIP3 (which allows necrosome formation), and elevated RIP1 kinase-dependent accumulation of reactive oxygen species (ROS). In conclusion, our data indicate that cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent ROS production.     

An IAP-IAP complex inhibits apoptosis

Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP(-/-) cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.     

Lipopolysaccharide induces the expression of cellular inhibitor of apoptosis protein-2 in human macrophages

Apoptosis is an important process in normal animal development as well as in diseases, and inhibitor of apoptosis protein (IAP) is one of the important factors that regulate apoptotic cell death. We found that lipopolysaccharide (LPS) enhances the expression of mRNA and protein of cellular IAP-2 (cIAP2) in human monoblastic U937 cells differentiated by phorbol ester pretreatment. cIAP2 mRNA was not detected in undifferentiated U937 cells. mRNAs of cIAP1 and X-chromosome-linked IAP (XIAP) were expressed constitutively and not affected by LPS in both undifferentiated and differentiated cells. LPS stimulated the expression of cIAP2 mRNA and protein in time- and concentration-dependent manners. LPS enhanced the expression of cIAP2 mRNA and protein in human monocyte-derived macrophages, which was associated with the inhibition of the caspase-3 activation, i.e., decrease in active p17 fragment of caspase-3 with simultaneous accumulation of precursor p20 fragment. We conclude that LPS may inhibit apoptosis of macrophages, at least in part, through the induction of cIAP2.     

The RING domain of cIAP1 mediates the degradation of RING-bearing inhibitor of apoptosis proteins by distinct pathways

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR-mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.     

The involvement of caspase-11 in TPEN-induced apoptosis

The depletion of intracellular zinc with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induces protein synthesis-dependent apoptosis. Here we examined the involvement of caspase induction in apoptosis. Among the examined caspases, only caspase-11 was increased by TPEN. Caspase-11 activity also increased, which resulted in caspase-3 activation. Cycloheximide or actinomycin D blocked caspase-11 induction, reduced caspase-11 and -3 activation, and attenuated TPEN-induced neuronal apoptosis. Blockade of caspase-11 by a chemical inhibitor or genetic deletion attenuated TPEN-induced apoptosis, indicating a critical role of caspase-11 in TPEN-induced apoptosis. Although mitochondria-mediated caspase-9/-3 activation also contributed to TPEN-induced apoptosis, caspase-11 is likely a key inducible apoptosis-inducing protein.     

Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes

We investigated the ability of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to reduce pancreatic cancer cell viability. TPEN was much more efficient to inhibit pancreatic adenocarcinoma cell growth than a panel of anti-cancer drugs, including 5-fluorouracil, irinotecan, cisplatin, edelfosine, trichostatin A, mitomycin C, and gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Moreover, TPEN showed a dose- and time-dependent anti-proliferative effect significantly higher on pancreatic cancer cells than on normal primary fibroblasts. This effect may be explained by a significantly higher zinc depletion by TPEN in pancreatic cancer cells as compared to fibroblasts. Cell viability reduction by TPEN was associated to both G1-phase cell cycle arrest and apoptosis, and to the increased ratio of the expression level of cyclin-Cdk inhibitor versus cyclin genes and apoptotic versus anti-apoptotic genes. Finally, we show that apoptotic cell death induced by TPEN involved mitochondrial injury and caspase 3 and caspase 8 activation. In this study, we suggest that zinc depletion may be an efficient strategy in the treatment of pancreatic cancer because of its reduced antiproliferative effect on normal cells.     

Microenvironment mesenchymal cells protect ovarian cancer cell lines from apoptosis by inhibiting XIAP inactivation

Epithelial ovarian carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. Carcinoma-associated mesenchymal stem cells (CA-MSC) have been shown to induce ovarian cancer chemoresistance through trogocytosis. Here we examined CA-MSC properties to protect ovarian cancer cells from carboplatin-induced apoptosis. Apoptosis was determined by Propidium Iodide and Annexin-V-FITC labelling and poly-ADP-ribose polymerase cleavage analysis. We showed a significant increase of inhibitory concentration 50 and a 30% decrease of carboplatin-induced apoptosis in ovarian cancer cells incubated in the presence of CA-MSC-conditioned medium (CM). A molecular analysis of apoptosis signalling pathway in response to carboplatin revealed that the presence of CA-MSC CM induced a 30% decrease of effector caspases-3 and -7 activation and proteolysis activity. CA-MSC secretions promoted Akt and X-linked inhibitor of apoptosis protein (XIAP; caspase inhibitor from inhibitor of apoptosis protein (IAP) family) phosphorylation. XIAP depletion by siRNA strategy permitted to restore apoptosis in ovarian cancer cells stimulated by CA-MSC CM. The factors secreted by CA-MSC are able to confer chemoresistance to carboplatin in ovarian cancer cells through the inhibition of effector caspases activation and apoptosis blockade. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and the phosphorylation of its downstream target XIAP underlined the implication of this signalling pathway in ovarian cancer chemoresistance. This study reveals the potentialities of targeting XIAP in ovarian cancer therapy.     

Degradation of survivin by the X-linked inhibitor of apoptosis (XIAP)-XAF1 complex

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a putative tumor suppressor in which expression is significantly reduced in human cancer cell lines and primary tumors. The proapoptotic effects of XAF1 have been attributed to both caspase-dependent and -independent means. In particular, XAF1 reverses the anti-caspase activity of XIAP, a physiological inhibitor of apoptosis. We further investigated the function of XAF1 by examining its relationship with other IAPs. Immunoprecipitation studies indicate that XAF1 binds to XIAP, cIAP1, cIAP2, Livin, TsIAP, and NAIP but not Survivin, an IAP that prevents mitotic catastrophe and in which antiapoptotic activity is exerted through direct XIAP interaction and stabilization. We found that overexpressed XAF1 down-regulates the protein expression of Survivin. Under these conditions, Survivin expression was restored in the presence of the proteasome inhibitor MG132 or a XIAP RING mutant that is defective in ubiquitin-protein isopeptide ligase (E3) activity, suggesting that XAF1 interaction activates E3 activity of XIAP and targets Survivin by direct ubiquitination. In addition, RNA interference targeting endogenous XIAP protected Survivin degradation by XAF1. Furthermore, interferon-beta-mediated XAF1 induction promoted formation of an endogenous XIAP-XAF1-Survivin complex. This complex facilitated Survivin degradation, which was prevented in XAF1(-/-) stable clones. Altogether, our study demonstrates that XAF1 mediates Survivin down-regulation through a complex containing XIAP, supporting dual roles for XAF1 in apoptosis and mitotic catastrophe.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

NF-kappaB-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion

Apoptosis plays a crucial role in maintenance of intestinal epithelial integrity and is highly regulated by numerous factors, including cellular polyamines. We recently showed that polyamines regulate nuclear factor (NF)-kappaB activity in normal intestinal epithelial (IEC-6) cells and that polyamine depletion activates NF-kappaB and promotes resistance to apoptosis. The current study went further to determine whether the inhibitors of apoptosis (IAP) family of proteins, c-IAP2 and XIAP, are downstream targets of activated NF-kappaB and play a role in antiapoptotic activity of polyamine depletion in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine not only activated NF-kappaB activity but also increased expression of c-IAP2 and XIAP. Specific inhibition of NF-kappaB by the recombinant adenoviral vector containing IkappaBalpha superrepressor (AdIkappaBSR) prevented the induction of c-IAP2 and XIAP in polyamine-deficient cells. Decreased levels of c-IAP2 and XIAP proteins by inactivation of NF-kappaB through AdIkappaBSR infection or treatment with the specific inhibitor Smac also overcame the resistance of polyamine-depleted cells to apoptosis induced by the combination of tumor necrosis factor (TNF)-alpha and cycloheximide (CHX). Although polyamine depletion did not alter levels of procaspase-3 protein, it inhibited formation of the active caspase-3. Decreased levels of c-IAP2 and XIAP by Smac prevented the inhibitory effect of polyamine depletion on the cleavage of procaspase-3 to the active caspase-3. These results indicate that polyamine depletion increases expression of c-IAP2 and XIAP by activating NF-kappaB in intestinal epithelial cells. Increased c-IAP2 and XIAP after polyamine depletion induce the resistance to TNF-alpha/CHX-induced apoptosis, at least partially, through inhibition of the caspase-3 activity.     

cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination

The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.     

The susceptibility to Fas-induced apoptosis in normal enterocytes is regulated on the level of cIAP1 and 2.

Various members of the tumor necrosis factor receptor superfamily are implicated in the regulation of enterocyte apoptosis. Previously, we observed that human intestinal epithelial cells are rather unsusceptible to Fas-induced apoptosis. In the present study we analyzed these protective mechanisms in the human intestinal epithelial cell line HIEC, focusing on antiapoptotic molecules of the IAP family which block apoptosis at the level of the caspase cascade. HIEC expressed all key molecules required to trigger Fas-induced apoptosis. However, no apoptosis occurred after activation of Fas, whereas an upregulation of antiapoptotic cIAP1 and 2 was observed. Suppression of this upregulation with the proteasome inhibitor MG132 or the protein synthesis inhibitor cycloheximide highly sensitized HIEC toward Fas-induced apoptosis. Western blot analyses revealed that both inhibitors potently suppressed endogenously produced cIAP1 and 2. No effect was observed on XIAP expression. These data indicate that enterocytes are particularly protected against Fas-induced apoptosis on the level of executionary caspases.