Novel molecular mechanism driving neuroprotection after soluble epoxide hydrolase inhibition: Insights for Alzheimer's disease therapeutics.

BACKGROUND: Neuroinflammation is widely recognized as a significant hallmark of Alzheimer's disease (AD). To combat neuroinflammation, the inhibition of the soluble epoxide hydrolase (sEH) enzyme has been demonstrated crucial. Importantly, sEH inhibition could be related to other neuroprotective pathways described in AD. AIMS: The aim of the study was to unveil new molecular pathways driving neuroprotection through sEH, we used an optimized, potent, and selective sEH inhibitor (sEHi, UB-SCG-51). MATERIALS AND METHODS: UB-SCG-51 was tested in neuroblastoma cell line, SH-SY5Y, in primary mouse and human astrocytes cultures challenged with proinflammatory insults and in microglia cultures treated with amyloid oligomers, as well as in mice AD model (5XFAD). RESULTS: UB-SCG-51 (10 and 30 µM) prevented neurotoxic reactive-astrocyte conversion in primary mouse astrocytes challenged with TNF-α, IL-1α, and C1q (T/I/C) combination for 24 h. Moreover, in microglial cultures, sEHi reduced inflammation and glial activity. In addition, UB-SCG-51 rescued 5XFAD cognitive impairment, reducing the number of Amyloid-ß plaques and Tau hyperphosphorylation accompanied by a reduction in neuroinflammation and apoptotic markers. Notably, a transcriptional profile analysis revealed a new pathway modulated by sEHi treatment. Specifically, the eIF2α/CHOP pathway, which promoted the endoplasmic reticulum response, was increased in the 5XFAD-treated group. These findings were confirmed in human primary astrocytes by combining sEHi and eIF2α inhibitor (eIF2αi) treatment. Besides, combining both treatments resulted in increased in C3 gene expression after T/I/C compared with the group treated with sEHi alone in cultures. DISCUSSION: Therefore, sEHi rescued cognitive impairment and neurodegeneration in AD mice model, based on the reduction of inflammation and eIF2α/CHOP signaling pathway. CONCLUSIONS: In whole, our results support the concept that targeting neuroinflammation through sEH inhibition is a promising therapeutic strategy to fight against Alzheimer's disease with additive and/or synergistic activities targeting neuroinflammation and cell stress.

A Novel, Selective c-Abl Inhibitor, Compound 5, Prevents Neurodegeneration in Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects movement. The nonreceptor tyrosine kinase c-Abl has shown a potential role in the progression of PD. As such, c-Abl inhibition is a promising candidate for neuroprotection in PD and α-synucleinopathies. Compound 5 is a newly synthesized blood-brain barrier penetrant c-Abl inhibitor with higher efficacy than existing inhibitors. The objective of the current study was to demonstrate the neuroprotective effects of compound 5 on the α-synuclein preformed fibril (α-syn PFF) mouse model of PD. Compound 5 significantly reduced neurotoxicity, activation of c-Abl, and Lewy body pathology caused by α-syn PFF in cortical neurons. Additionally, compound 5 markedly ameliorated the loss of dopaminergic neurons, c-Abl activation, Lewy body pathology, neuroinflammatory responses, and behavioral deficits induced by α-syn PFF injection in vivo. Taken together, these results suggest that compound 5 could be a pharmaceutical agent to prevent the progression of PD and α-synucleinopathies.

Tyrosine kinase inhibitor neratinib attenuates liver fibrosis by targeting activated hepatic stellate cells.

Liver fibrosis, a common outcome of chronic liver disease characterized by excessive accumulation of extracellular matrix (ECM), is a leading cause of mortality worldwide. The tyrosine kinase inhibitor neratinib is a human epidermal growth factor receptor 2 (HER2) inhibitor approved by the FDA for HER2-positive breast cancer treatment; however, it has not yet been evaluated for liver fibrosis treatment. We elucidated the anti-fibrotic effects of neratinib in hepatic stellate cells (HSCs) and in vivo models of CCl4-induced liver fibrosis. HSC activation is a key step in liver fibrogenesis and has a crucial role in collagen deposition, as it is primarily responsible for excessive ECM production. The effect of neratinib on HSC was evaluated in transforming growth factor (TGF-ß)-incubated LX-2 cells and culture-activated primary human HSCs. In vivo study results indicated that neratinib inhibited the inflammatory response, HSC differentiation, and collagen accumulation induced by CCl4. Moreover, the anti-fibrotic effects of neratinib were not associated with the HER2 signaling pathways. Neratinib inhibited FGF2 expression in activated HSCs and serum FGF2 level in the model, suggesting that neratinib possessed therapeutic potency against liver fibrosis and the potential for application against other fibrotic diseases.

DR4-Ser424 O-GlcNAcylation Promotes Sensitization of TRAIL-Tolerant Persisters and TRAIL-Resistant Cancer Cells to Death.

TNF-related apoptosis-inducing ligand (TRAIL) resistance, including nongenetically acquired tolerance in cancer persister cells, is a major obstacle to translating TRAIL therapy into patients with cancer. However, the underlying mechanisms remain to be elucidated. Here, we show that DR4/TRAIL-R1 is O-GlcNAcylated at Ser424 in its death domain to mediate both apoptosis and necrosis upon TRAIL ligation. We found that DR4-Ser424 mutations, identified from our cell-based functional screen using a cancer patient-derived cDNA expression library and from The Cancer Genome Atlas, caused TRAIL resistance in various human cancer cell lines. Using O-GlcNAc transferase knockdown cells, DR4-preferred versus DR5-preferred cancer cells, and a DR5-neutralizing antibody, we evaluated the essential role of DR4-specific O-GlcNAc modification in TRAIL cytotoxicity. In contrast to DR4, DR5 was not O-GlcNAcylated by TRAIL treatment, discriminating DR4 from DR5-mediated signaling. Apart from genetic changes in DR4-Ser424, we further classified various cancer cell lines originated from stomach, colon, lung, and glioblastoma according to their sensitivity to and receptor preference upon TRAIL death signaling and generated TRAIL-tolerant persister-derived DLD-1PER cells. Among these, we discovered that DR4 was not modified by O-GlcNAc in most of the TRAIL-resistant cancer cells and DLD-1PER cells. Interestingly, promoting DR4 O-GlcNAcylation intentionally using 2-deoxy-d-glucose or a high concentration of glucose sensitized those resistant cancer cells to TRAIL. The O-GlcNAcylation-defective DR4 failed to form DISC/necrosome and could not translocate to aggregated platforms for receptor clustering. Our findings demonstrate that DR4 O-GlcNAcylation is crucial for TRAIL death signaling, providing new opportunities for TRAIL therapy overcoming TRAIL resistance in cancers. SIGNIFICANCE: This study reports that a novel posttranslational modification by O-GlcNAcylation of one of the two human TRAIL receptors with a death domain, TRAIL-R1 (DR4), plays a crucial role in enabling both apoptotic and necroptotic cell death induction by TRAIL.

Targeting of dermal myofibroblasts through death receptor 5 arrests fibrosis in mouse models of scleroderma.

Scleroderma is an autoimmune rheumatic disorder accompanied by severe fibrosis in skin and other internal organs. During scleroderma progression, resident fibroblasts undergo activation and convert to α-smooth muscle actin (α-SMA) expressing myofibroblasts (MFBs) with increased capacity to synthesize collagens and fibrogenic components. Accordingly, MFBs are a major therapeutic target for fibrosis in scleroderma and treatment with blocking MFBs could produce anti-fibrotic effects. TLY012 is an engineered human TNF-related apoptosis-inducing ligand (TRAIL) which induces selective apoptosis in transformed cells expressing its cognate death receptors (DRs). Here we report that TLY012 selectively blocks activation of dermal fibroblasts and induces DR-mediated apoptosis in α-SMA+ MFBs through upregulated DR5 during its activation. In vivo, TLY012 reverses established skin fibrosis to near-normal skin architecture in mouse models of scleroderma. Thus, the TRAIL pathway plays a critical role in tissue remodeling and targeting upregulated DR5 in α-SMA+ MFBs is a viable therapy for fibrosis in scleroderma.

Hypoxia-tropic Protein Nanocages for Modulation of Tumor- and Chemotherapy-Associated Hypoxia.

Despite its central role in tumor progression and treatment resistance, poor vascularization that necessitates penetration of therapeutics through tumor extracellular matrix (ECM) constitutes a significant challenge to managing tumor hypoxia via conventional systemic treatment regimens. In addition, methods to target hypoxic tumor cells are lacking. Here, we discovered that human ferritin nanocages (FTn) possess an intrinsic ability to preferentially engage with hypoxic tumor tissues, in addition to normoxic tumor areas. We also developed a simple method of endowing FTn with spatially controlled "mosaic" surface poly(ethylene glycol) (PEG) coatings that facilitate deep penetration of FTn through ECM to reach hypoxic tumor tissues while retaining its inherent hypoxia-tropic property. Hypoxia-inhibiting agents systemically delivered via this surface-PEGylated FTn were readily accumulated in hypoxic tumor tissues, thereby providing significantly enhanced therapeutic benefits compared to the identical agents delivered in solution as a stand-alone therapy or an adjuvant to restore efficacy of conventional systemic chemotherapy.

Inhibition of euchromatin histone-lysine N-methyltransferase 2 sensitizes breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand through reactive oxygen species-mediated activating transcription factor 4-C/EBP homologous protein-death receptor 5 pathway activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been characterized as an anti-cancer therapeutic agent with prominent cancer cell selectivity over normal cells. However, breast cancer cells are generally resistant to TRAIL, thus limiting its therapeutic potential. In this study, we found that BIX-01294, a selective inhibitor of euchromatin histone methyltransferase 2/G9a, is a strong TRAIL sensitizer in breast cancer cells. The combination of BIX-01294 and TRAIL decreased cell viability and led to an increase in the annexin V/propidium iodide-positive cell population, DNA fragmentation, and caspase activation. BIX-01294 markedly increased death receptor 5 (DR5) expression, while silencing of DR5 using small interfering RNAs abolished the TRAIL-sensitizing effect of BIX-01294. Specifically, BIX-01294 induced C/EBP homologous protein (CHOP)-mediated DR5 gene transcriptional activation and DR5 promoter activation was induced by upregulation of the protein kinase R-like endoplasmic reticulum kinase-mediated activating transcription factor 4 (ATF4). Moreover, inhibition of reactive oxygen species by N-acetyl-L-cysteine efficiently blocked BIX-01294-induced DR5 upregulation by inhibiting ATF4/CHOP expression, leading to diminished sensitization to TRAIL. These findings suggest that BIX-01294 sensitizes breast cancer cells to TRAIL by upregulating ATF4/CHOP-dependent DR5 expression with a reactive oxygen species-dependent manner. Furthermore, combination treatment with BIX-01294 and TRAIL suppressed tumor growth and induced apoptosis in vivo. In conclusion, we found that epigenetic regulation can contribute to the development of resistance to cancer therapeutics such as TRAIL, and further studies of unfolded protein responses and the associated epigenetic regulatory mechanisms may lead to the discovery of new molecular targets for effective cancer therapy.

PEGylated TRAIL ameliorates experimental inflammatory arthritis by regulation of Th17 cells and regulatory T cells.

TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand that can induce apoptosis in cells expressing its cognate death receptors (DRs). Previously, we demonstrated the therapeutic potential of recombinant human TRAIL in experimental rheumatoid arthritis (RA) models. However, the mechanisms of how DR-mediated apoptosis elicits these actions is not known. Here, we show that systemically administering a potent, long-acting PEGylated TRAIL (TRAILPEG) is profoundly anti-rheumatic against two complementary experimental RA mouse models, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA), via targeting IL-17 secreting Th17 cells and regulatory T cells (Treg). Systemic administration of TRAILPEG after disease onset ameliorated the severity of inflammatory arthritis including arthritis indices, paw thickness, cartilage damage and neutrophil infiltration in both CIA and CAIA models. Additionally, the levels of inflammatory molecules (p-p65, ICAM-1, Cox-2, MMP3, and iNOS), pro-inflammatory cytokines (TNF-α, IL-1ß, IFN-γ, IL-6, IL-17) and accumulation of activated macrophages were significantly reduced after the TRAILPEG treatment. Importantly, TRAILPEG decreased the number of pro-inflammatory Th17 cells in inflamed arthritic joints through TRAIL-induced apoptosis while increasing anti-inflammatory Treg population in vivo. These results suggest that TRAILPEG ameliorates autoimmunity by targeting the Th 17-Tregs axis, making it a promising candidate drug for the treatment of RA.

ENC1 Modulates the Aggregation and Neurotoxicity of Mutant Huntingtin Through p62 Under ER Stress.

Huntington's disease (HD) is a devastating neurodegenerative disorder, which is caused by the expression and aggregation of polyQ-expanded mutant huntingtin protein (mtHTT). While toxic mtHTT aggregates are primarily eliminated through autophagy, autophagy dysfunction is often observed in HD pathogenesis. Here, we show that ectodermal-neural cortex 1 (ENC1), a novel binding partner of sequestosome 1 (p62), negatively regulates autophagy under endoplasmic reticulum (ER) stress. We found that ER stress significantly increases the expression of ENC1 via inositol-requiring enzyme 1 (IRE1)-TNF receptor-associated factor 2 (TRAF2)-c-Jun N-terminal kinase (JNK) pathway. Ectopic expression of ENC1 alone induces the accumulation of detergent-resistant mtHTT aggregates and downregulation of ENC1 alleviates ER stress-induced mtHTT aggregation. Simultaneously, ER stress-induced impairment of autophagy flux is ameliorated by downregulation of ENC1. From immunoprecipitation and immunocytochemical assays, we found that ENC1 binds to p62 through its BTB and C-terminal Kelch (BACK) domain and this interaction is enhanced under ER stress. In particular, ENC1 preferentially interacts with the phosphorylated p62 at Ser403 during ER stress. Interestingly, ENC1 colocalizes with mtHTT aggregates and its C-terminal Kelch domain is required for interfering with the access of p62 to ubiquitinated mtHTT aggregates, thus inhibiting cargo recognition of p62. Accordingly, knockdown of ENC1 expression enhances colocalization of p62 with mtHTT aggregates. Consequently, ENC1 knockdown relieves death of neuronal cells expressing mtHTT under ER stress. These results suggest that ENC1 interacts with the phosphorylated p62 to impair autophagic degradation of mtHTT aggregates and affects cargo recognition failure under ER stress, leading to the accumulation and neurotoxicity of mtHTT aggregates.

Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells.

UNLABELLED: Liver fibrosis is a common outcome of chronic liver disease that leads to liver cirrhosis and hepatocellular carcinoma. No US Food and Drug Administration-approved targeted antifibrotic therapy exists. Activated hepatic stellate cells (aHSCs) are the major cell types responsible for liver fibrosis; therefore, eradication of aHSCs, while preserving quiescent HSCs and other normal cells, is a logical strategy to stop and/or reverse liver fibrogenesis/fibrosis. However, there are no effective approaches to specifically deplete aHSCs during fibrosis without systemic toxicity. aHSCs are associated with elevated expression of death receptors and become sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. Treatment with recombinant TRAIL could be a potential strategy to ameliorate liver fibrosis; however, the therapeutic application of recombinant TRAIL is halted due to its very short half-life. To overcome this problem, we previously generated PEGylated TRAIL (TRAILPEG ) that has a much longer half-life in rodents than native-type TRAIL. In this study, we demonstrate that intravenous TRAILPEG has a markedly extended half-life over native-type TRAIL in nonhuman primates and has no toxicity in primary human hepatocytes. Intravenous injection of TRAILPEG directly induces apoptosis of aHSCs in vivo and ameliorates carbon tetrachloride-induced fibrosis/cirrhosis in rats by simultaneously down-regulating multiple key fibrotic markers that are associated with aHSCs. CONCLUSION: TRAIL-based therapies could serve as new therapeutics for liver fibrosis/cirrhosis and possibly other fibrotic diseases. (Hepatology 2016;64:209-223).

Delivery of tumor-homing TRAIL sensitizer with long-acting TRAIL as a therapy for TRAIL-resistant tumors.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has attracted great interest as a cancer therapy because it selectively induces death receptor (DR)-mediated apoptosis in cancer cells while sparing normal tissue. However, recombinant human TRAIL demonstrates limited therapeutic efficacy in clinical trials, possibly due to TRAIL-resistance of primary cancers and its inherent short half-life. Here we introduce drug delivery approaches to maximize in vivo potency of TRAIL in TRAIL-resistant tumor xenografts by (1) extending the half-life of the ligand with PEGylated TRAIL (TRAILPEG) and (2) concentrating a TRAIL sensitizer, selected from in vitro screening, in tumors via tumor-homing nanoparticles. Antitumor efficacy of TRAILPEG with tumor-homing sensitizer was evaluated in HCT116 and HT-29 colon xenografts. Western blot, real-time PCR, immunohistochemistry and cell viability assays were employed to investigate mechanisms of action and antitumor efficacy of the combination. We discovered that doxorubicin (DOX) sensitizes TRAIL-resistant HT-29 colon cancer cells to TRAIL by upregulating mRNA expression of DR5 by 60% in vitro. Intravenously administered free DOX does not effectively upregulate DR5 in tumor tissues nor demonstrate synergy with TRAILPEG in HT-29 xenografts, but rather introduces significant systemic toxicity. Alternatively, when DOX was encapsulated in hyaluronic acid-based nanoparticles (HAC/DOX) and intravenously administered with TRAILPEG, DR-mediated apoptosis was potentiated in HT-29 tumors by upregulating DR5 protein expression by 70% and initiating both extrinsic and intrinsic apoptotic pathways with reduced systemic toxicity compared to HAC/DOX or free DOX combined with TRAILPEG (80% vs. 40% survival rate; 75% vs. 34% tumor growth inhibition). This study demonstrates a unique approach to overcome TRAIL-based therapy drawbacks using sequential administration of a tumor-homing TRAIL sensitizer and long-acting TRAILPEG.

Highly PEGylated DNA Nanoparticles Provide Uniform and Widespread Gene Transfer in the Brain.

Gene delivery to the central nervous system (CNS) has potential as a means for treating numerous debilitating neurological diseases. Nonviral gene vector platforms are tailorable and can overcome key limitations intrinsic to virus-mediated delivery; however, lack of clinical efficacy with nonviral systems to date may be attributed to limited gene vector dispersion and transfection in vivo. It is shown that the brain extracellular matrix (ECM) strongly limits penetration of polymer-based gene vector nanoparticles (NP) through the brain parenchyma, even when they are very small (<60 nm) and coated with a polyethylene glycol (PEG) corona of typical density. Following convection enhanced delivery (CED), conventional gene vectors are confined to the injection site, presumably by adhesive interactions with the brain ECM and do not provide gene expression beyond the point of administration. In contrast, it is found that incorporating highly PEGylated polymers allows the production of compacted (≈43 nm) and colloidally stable DNA NP that avoid adhesive trapping within the brain parenchyma. When administered by CED into the rat striatum, highly PEGylated DNA NP distribute throughout and provide broad transgene expression without vector-induced toxicity. The use of these brain-penetrating gene vectors, in conjunction with CED, offers an avenue to improve gene therapy for CNS diseases.

Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells.

Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (∼ 21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ∼ 100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications.

The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth.

Adenylate kinase 2 (AK2), which balances adenine nucleotide pool, is a multi-functional protein. Here we show that AK2 negatively regulates tumour cell growth. AK2 forms a complex with dual-specificity phosphatase 26 (DUSP26) phosphatase and stimulates DUSP26 activity independently of its AK activity. AK2/DUSP26 phosphatase protein complex dephosphorylates fas-associated protein with death domain (FADD) and regulates cell growth. AK2 deficiency enhances cell proliferation and induces tumour formation in a xenograft assay. This anti-growth function of AK2 is associated with its DUSP26-stimulating activity. Downregulation of AK2 is frequently found in tumour cells and human cancer tissues showing high levels of phospho-FADD(Ser194). Moreover, reconstitution of AK2 in AK2-deficient tumour cells retards both cell proliferation and tumourigenesis. Consistent with this, AK2(+/-) mouse embryo fibroblasts exhibit enhanced cell proliferation with a significant alteration in phospho-FADD(Ser191). These results suggest that AK2 is an associated activator of DUSP26 and suppresses cell proliferation by FADD dephosphorylation, postulating AK2 as a negative regulator of tumour growth.

Intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53.

Osteopontin (OPN) is highly expressed in cancer patients and plays important roles in many stages of tumor progression, such as anti-apoptosis, proliferation, and metastasis. From functional screening of human cDNA library, we isolated OPN as a caspase-8 substrate that regulates cell death during hypoxia/reoxygenation (Hyp/RO). In vitro cleavage assays demonstrate that OPN is cleaved at Asp-135 and Asp-157 by caspase-8. Cellular cleavage of OPN is observed in apoptotic cells exposed to Hyp/RO among various apoptotic stimuli and its cleavage is blocked by zVAD or IETD caspase inhibitor. Further, over-expression of OPN, the form with secretion signal, inhibits Hyp/RO-induced cell death. Caspase cleavage-defective OPN mutant (OPN D135A/D157A) is more efficient to suppress Hyp/RO-induced cell death than wild-type OPN. OPN D135A/D157A sustains AKT activity to increase cell viability through inhibition of caspase-9 during Hyp/RO. In addition, OPN is highly induced in some tumor cells during Hyp/RO, such as HeLa and Huh-7 cells, which is associated with their resistance to Hyp/RO by sustaining AKT activity. Notably, OPN C-terminal cleavage fragment produced by caspase-8 is detected in the nucleus. Plasmid-encoded expression of OPN C-terminal cleavage fragment increases p53 protein level and induces apoptosis of wild-type mouse embryonic fibroblast cells, but not p53(-/-) mouse embryonic fibroblast cells. These observations suggest that the protective function of OPN during Hyp/RO is inactivated via the proteolytic cleavage by caspase-8 and its cleavage product subsequently induces cell death via p53, postulating caspase-8 as a negative regulator of tumorigenic activity of OPN.

AK2 activates a novel apoptotic pathway through formation of a complex with FADD and caspase-10.

Mitochondrial proteins function as essential regulators in apoptosis. Here, we show that mitochondrial adenylate kinase 2 (AK2) mediates mitochondrial apoptosis through the formation of an AK2-FADD-caspase-10 (AFAC10) complex. Downregulation of AK2 attenuates etoposide- or staurosporine-induced apoptosis in human cells, but not that induced by tumour-necrosis-factor-related apoptosis-inducing ligand (TRAIL) or Fas ligand (FasL). During intrinsic apoptosis, AK2 translocates to the cytoplasm, whereas this event is diminished in Apaf-1 knockdown cells and prevented by Bcl-2 or Bcl-X(L). Addition of purified AK2 protein to cell extracts first induces activation of caspase-10 via FADD and subsequently caspase-3 activation, but does not affect caspase-8. AFAC10 complexes are detected in cells undergoing intrinsic cell death and AK2 promotes the association of caspase-10 with FADD. In contrast, AFAC10 complexes are not detected in several etoposide-resistant human tumour cell lines. Taken together, these results suggest that, acting in concert with FADD and caspase-10, AK2 mediates a novel intrinsic apoptotic pathway that may be involved in tumorigenesis.