Selective BH3-mimetics targeting BCL-2, BCL-XL or MCL-1 induce severe mitochondrial perturbations.

Induction of apoptosis by selective BH3-mimetics is currently investigated as a novel strategy for cancer treatment. Here, we report that selective BH3-mimetics induce apoptosis in a variety of hematological malignancies. Apoptosis is accompanied by severe mitochondrial toxicities upstream of caspase activation. Specifically, the selective BH3-mimetics ABT-199, A-1331852 and S63845, which target BCL-2, BCL-XL and MCL-1, respectively, induce comparable ultrastructural changes including mitochondrial swelling, a decrease of mitochondrial matrix density and severe loss of cristae structure. These shared effects on mitochondrial morphology indicate a similar function of these anti-apoptotic BCL-2 proteins in maintaining mitochondrial integrity and function.

Targeting intermediary metabolism enhances the efficacy of BH3 mimetic therapy in hematologic malignancies.

BH3 mimetics are novel targeted drugs with remarkable specificity, potency and enormous potential to improve cancer therapy. However, acquired resistance is an emerging problem. We report the rapid development of resistance in chronic lymphocytic leukemia cells isolated from patients exposed to increasing doses of navitoclax (ABT-263), a BH3 mimetic. To mimic such rapid development of chemoresistance, we developed simple resistance models to three different BH3 mimetics, targeting BCL-2 (ABT-199), BCL-XL (A-1331852) or MCL-1 (A-1210477), in relevant hematologic cancer cell lines. In these models, resistance could not be attributed to either consistent changes in expression levels of the anti-apoptotic proteins or interactions among different pro- and anti-apoptotic BCL-2 family members. Using genetic silencing, pharmacological inhibition and metabolic supplementation, we found that targeting glutamine uptake and its downstream signaling pathways, namely glutaminolysis, reductive carboxylation, lipogenesis, cholesterogenesis and mammalian target of rapamycin signaling resulted in marked sensitization of the chemoresistant cells to BH3 mimetic-mediated apoptosis. Furthermore, our findings highlight the possibility of repurposing widely used drugs, such as statins, to target intermediary metabolism and improve the efficacy of BH3 mimetic therapy.

Reconstitution of the death-inducing signaling complex reveals a substrate switch that determines CD95-mediated death or survival.

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

BH3 profiling and a toolkit of BH3-mimetic drugs predict anti-apoptotic dependence of cancer cells.

BACKGROUND: Anti-apoptotic BCL-2 family members antagonise apoptosis by sequestering their pro-apoptotic counterparts. The balance between the different BCL-2 family members forms the basis of BH3 profiling, a peptide-based technique used to predict chemosensitivity of cancer cells. Recent identification of cell-permeable, selective inhibitors of BCL-2, BCL-XL and MCL-1, further facilitates the determination of the BCL-2 family dependency of cancer cells. METHODS: We use BH3 profiling in combination with cell death analyses using a chemical inhibitor toolkit to assess chemosensitivity of cancer cells. RESULTS: Both BH3 profiling and the inhibitor toolkit effectively predict chemosensitivity of cells addicted to a single anti-apoptotic protein but a combination of both techniques is more instructive when cell survival depends on more than one anti-apoptotic protein. CONCLUSIONS: The inhibitor toolkit provides a rapid, inexpensive and simple means to assess the chemosensitivity of tumour cells and in conjunction with BH3 profiling offers much potential in personalising cancer therapy.

BCL2/BCL-X(L) inhibition induces apoptosis, disrupts cellular calcium homeostasis, and prevents platelet activation.

Apoptosis in megakaryocytes results in the formation of platelets. The role of apoptotic pathways in platelet turnover and in the apoptotic-like changes seen after platelet activation is poorly understood. ABT-263 (Navitoclax), a specific inhibitor of antiapoptotic BCL2 proteins, which is currently being evaluated in clinical trials for the treatment of leukemia and other malignancies, induces a dose-limiting thrombocytopenia. In this study, the relationship between BCL2/BCL-X(L) inhibition, apoptosis, and platelet activation was investigated. Exposure to ABT-263 induced apoptosis but repressed platelet activation by physiologic agonists. Notably, ABT-263 induced an immediate calcium response in platelets and the depletion of intracellular calcium stores, indicating that on BCL2/BCL-X(L) inhibition platelet activation is abrogated because of a diminished calcium signaling. By comparing the effects of ABT-263 and its analog ABT-737 on platelets and leukemia cells from the same donor, we show, for the first time, that these BCL2/BCL-X(L) inhibitors do not offer any selective toxicity but induce apoptosis at similar concentrations in leukemia cells and platelets. However, reticulated platelets are less sensitive to apoptosis, supporting the hypothesis that treatment with ABT-263 induces a selective loss of older platelets and providing an explanation for the transient thrombocytopenia observed on ABT-263 treatment.

Novel roles of RTN4 and CLIMP-63 in regulating mitochondrial structure, bioenergetics and apoptosis.

The recruitment of DRP1 to mitochondrial membranes prior to fission is facilitated by the wrapping of endoplasmic reticulum (ER) membranes around the mitochondria. To investigate the complex interplay between the ER membranes and DRP1 in the context of mitochondrial structure and function, we downregulate two key ER shaping proteins, RTN4 and CLIMP-63, and demonstrate pronounced mitochondrial hyperfusion and reduced ER-mitochondria contacts, despite their differential regulation of ER architecture. Although mitochondrial recruitment of DRP1 is unaltered in cells lacking RTN4 or CLIMP-63, several aspects of mitochondrial function, such as mtDNA-encoded translation, respiratory capacity and apoptosis are significantly hampered. Further mechanistic studies reveal that CLIMP-63 is required for cristae remodeling (OPA1 proteolysis) and DRP1-mediated mitochondrial fission, whereas both RTN4 and CLIMP-63 regulate the recruitment of BAX to ER and mitochondrial membranes to enable cytochrome c release and apoptosis, thereby performing novel and distinct roles in the regulation of mitochondrial structure and function.

The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein.

Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using (3)H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia.

ABT-737 and its orally active analog, ABT-263, are rationally designed inhibitors of BCL2 and BCL-X(L). ABT-263 shows promising activity in early phase 1 clinical trials in B-cell malignancies, particularly chronic lymphocytic leukemia (CLL). In vitro, peripheral blood CLL cells are extremely sensitive to ABT-737 (EC(50) approximately 7 nM), with rapid induction of apoptosis in all 60 patients tested, independent of parameters associated with disease progression and chemotherapy resistance. In contrast to data from cell lines, ABT-737-induced apoptosis in CLL cells was largely MCL1-independent. Because CLL cells within lymph nodes are more resistant to apoptosis than those in peripheral blood, CLL cells were cultured on CD154-expressing fibroblasts in the presence of interleukin-4 (IL-4) to mimic the lymph node microenvironment. CLL cells thus cultured developed an approximately 1000-fold resistance to ABT-737 within 24 hours. Investigations of the underlying mechanism revealed that this resistance occurred upstream of mitochondrial perturbation and involved de novo synthesis of the antiapoptotic proteins BCL-X(L) and BCL2A1, which were responsible for resistance to low and high ABT-737 concentrations, respectively. Our data indicate that after therapy with ABT-737-related inhibitors, resistant CLL cells might develop in lymph nodes in vivo and that treatment strategies targeting multiple BCL2 antiapoptotic members simultaneously may have synergistic activity.

Role of NOXA and its ubiquitination in proteasome inhibitor-induced apoptosis in chronic lymphocytic leukemia cells.

BACKGROUND: Bortezomib has been successfully used in the treatment of multiple myeloma and has been proposed as a potential treatment for chronic lymphocytic leukemia. In this study we investigated the mechanism by which bortezomib induces apoptosis in chronic lymphocytic leukemia cells. DESIGN AND METHODS: Using western blot analysis, we monitored the regulation of BCL2 family members, proteins of the unfolded protein response (endoplasmic reticulum stress response) and activation of caspases in relation to induction of apoptosis (measured by annexin-propidium iodide staining and loss of mitochondrial membrane potential) by bortezomib in chronic lymphocytic leukemia cells. RESULTS: Bortezomib induced apoptosis through activation of the mitochondrial pathway independently of changes associated with endoplasmic reticulum stress. Perturbation of mitochondria was regulated by a rapid and transcription-independent increase of NOXA protein, which preceded release of cytochrome c, HtrA2, Smac and activation of caspase-9 and -3. NOXA had a short half life (approximately 1-2 h) and was ubiquitinated on at least three primary lysine residues, resulting in proteasomal-dependent degradation. Down-regulation of NOXA, using short interfering RNA in chronic lymphocytic leukemia cells, decreased bortezomib-induced apoptosis. Finally bortezomib when combined with seliciclib resulted in a stronger and earlier increase in NOXA protein, caspase-3 cleavage and induction of apoptosis in chronic lymphocytic leukemia cells. CONCLUSIONS: These results highlight a critical role for NOXA in bortezomib-induced apoptosis in chronic lymphocytic leukemia cells and suggest that this drug may become more efficient for the treatment of chronic lymphocytic leukemia if combined with other agents able to interfere with the basal levels of MCL1.

Phenylarsine oxide interferes with the death inducing signaling complex and inhibits tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis.

The mechanism by which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death is the subject of intense scrutiny due to its preferential targeting of transformed cells for deletion. Based on recent findings that the TRAIL-dependent death inducing signaling complex (DISC) forms and signals at the plasma membrane without being internalized, we investigated the possibility that agents that prevent endocytosis may stabilize the surface bound DISC and thereby enhance TRAIL-dependent signaling. We utilized phenylarsine oxide (PAO), a trivalent arsenical that has been reported to inhibit endocytosis and to induce mitochondrial permeability transition. Therefore PAO could, by two separate and independent activities, enhance TRAIL-induced killing. Paradoxically, we found that rather than synergizing with TRAIL, PAO was an effective inhibitor of TRAIL-induced killing. Recruitment of FADD and caspase-8 to the TRAIL-dependent DISC was diminished in a concentration-dependent manner in cells exposed to PAO. The effects of PAO could not be reversed by washing cells under non-reducing conditions, suggesting covalent linkage of PAO with its cellular target(s); however, 2,3-dimercaptoethanol effectively overcame the inhibitory action of PAO and restored sensitivity to TRAIL-induced apoptosis. PAO inhibited formation of the TRAIL-dependent DISC and therefore prevented all subsequent apoptotic events.

Cleavage of the transactivation-inhibitory domain of p63 by caspases enhances apoptosis.

p63 is a p53-related transcription factor. Utilization of two different promoters and alternative splicing at the C terminus lead to generation of six isoforms. The alpha isoforms of TAp63 and DeltaNp63 contain a transactivation-inhibitory (TI) domain at the C termini, which can bind to the transactivation (TA) domain and inhibit its transcriptional activity. Consequently, TAp63alpha can directly inhibit its activity through an intramolecular interaction; similarly, DeltaNp63alpha can inhibit the activity of the active TAp63 isoforms through an intermolecular interaction. In this work, we demonstrate that after induction of apoptosis, the TI domain of the p63alpha isoforms is cleaved by activated caspases. Cleavage of DeltaNp63alpha relieves its inhibitory effect on the transcriptionally active p63 proteins, and the cleavage of TAp63alpha results in production of a TAp63 protein with enhanced transcriptional activity. In agreement with these data, generation of the N-terminal TAp63 fragment has a role in apoptosis because stable cell lines expressing wild-type TAp63 are more sensitive to apoptosis compared with cells expressing the noncleavable mutant. We also used a model system in which TAp63 expression was induced by trichostatin-A treatment in HCT116 cells. Trichostatin-A sensitized these cells to apoptosis, and this sensitization was associated with cleavage of up-regulated p63.

Death receptor-induced apoptosis reveals a novel interplay between the chromosomal passenger complex and CENP-C during interphase.

Despite the fact that the chromosomal passenger complex is well known to regulate kinetochore behavior in mitosis, no functional link has yet been established between the complex and kinetochore structure. In addition, remarkably little is known about how the complex targets to centromeres. Here, in a study of caspase-8 activation during death receptor-induced apoptosis in MCF-7 cells, we have found that cleaved caspase-8 rapidly translocates to the nucleus and that this translocation is correlated with loss of the centromere protein (CENP)-C, resulting in extensive disruption of centromeres. Caspase-8 activates cytoplasmic caspase-7, which is likely to be the primary caspase responsible for cleavage of CENP-C and INCENP, a key chromosomal passenger protein. Caspase-mediated cleavage of CENP-C and INCENP results in their mislocalization and the subsequent mislocalization of Aurora B kinase. Our results demonstrate that the chromosomal passenger complex is displaced from centromeres as a result of caspase activation. Furthermore, mutation of the primary caspase cleavage sites of INCENP and CENP-C and expression of noncleavable CENP-C or INCENP prevent the mislocalization of the passenger complex after caspase activation. Our studies provide the first evidence for a functional interplay between the passenger complex and CENP-C.

Caspase cleavage of Itch in chronic lymphocytic leukemia cells.

E3 ubiquitin ligases catalyze the final step in the ubiquitylation cascade and therefore determine the specificity of this important cellular metabolic pathway. Although first thought to be constitutively active, increasing evidence demonstrates the existence of a wide variety of posttranslational modifications that regulate the activity of these enzymes. Here we show that upon induction of apoptosis the ubiquitin ligase Itch is processed by caspases both in vitro and ex vivo in cells from patients with chronic lymphocytic leukemia (CLL). The specific cleavage site was mapped to residue Asp240. Interestingly, cleavage of Itch by active caspases does not inhibit the catalytic activity of Itch, but results in the loss of an N-terminal Itch fragment that contains a negative regulatory region. Our data suggests that caspase-dependent Itch cleavage might be an important regulator of Itch at the endogenous level under both physiological and stressed conditions.

DEDD regulates degradation of intermediate filaments during apoptosis.

Apoptosis depends critically on regulated cytoskeletal reorganization events in a cell. We demonstrate that death effector domain containing DNA binding protein (DEDD), a highly conserved and ubiquitous death effector domain containing protein, exists predominantly as mono- or diubiquitinated, and that diubiquitinated DEDD interacts with both the K8/18 intermediate filament network and pro-caspase-3. Early in apoptosis, both cytosolic DEDD and its close homologue DEDD2 formed filaments that colocalized with and depended on K8/18 and active caspase-3. Subsequently, these filamentous structures collapsed into intracellular inclusions that migrated into cytoplasmic blebs and contained DEDD, DEDD2, active caspase-3, and caspase-3-cleaved K18 late in apoptosis. Biochemical studies further confirmed that DEDD coimmunoprecipitated with both K18 and pro-caspase-3, and kinetic analyses placed apoptotic DEDD staining prior to caspase-3 activation and K18 cleavage. In addition, both caspase-3 activation and K18 cleavage was inhibited by expression of DEDDDeltaNLS1-3, a cytosolic form of DEDD that cannot be ubiquitinated. Finally, siRNA mediated DEDD knockdown cells exhibited inhibition of staurosporine-induced DNA degradation. Our data suggest that DEDD represents a novel scaffold protein that directs the effector caspase-3 to certain substrates facilitating their ordered degradation during apoptosis.

Apogossypol-mediated reorganisation of the endoplasmic reticulum antagonises mitochondrial fission and apoptosis.

The endoplasmic reticulum (ER) with its elaborate network of highly curved tubules and flat sheets interacts with several other organelles, including mitochondria, peroxisomes and endosomes, to play vital roles in their membrane dynamics and functions. Previously, we identified structurally diverse chemicals from different pharmacological classes, which induce a reversible reorganisation of ER membranes. Using apogossypol as a prototypic tool compound, we now show that ER membrane reorganisation occurs at the level of ER tubules but does not involve ER sheets. Reorganisation of ER membranes prevents DRP-1-mediated mitochondrial fission, thereby antagonising the functions of several mitochondrial fission-inducing agents. Previous reports have suggested that ER membranes mark the constriction sites of mitochondria by localising DRP-1, as well as BAX on mitochondrial membranes to facilitate both mitochondrial fission and outer membrane permeabilisation. Following ER membrane reorganisation and subsequent exposure to an apoptotic stimulus (BH3 mimetics), DRP-1 still colocalises with the reorganised ER membranes but BAX translocation and activation, cytochrome c release and phosphatidylserine externalisation are all inhibited, thereby diminishing the ability of BH3 mimetics to induce the intrinsic apoptotic pathway. Strikingly, both ER membrane reorganisation and its resulting inhibition of apoptosis could be reversed by inhibitors of dihydroorotate dehydrogenase (DHODH), namely teriflunomide and its active metabolite, leflunomide. However, neither genetic inhibition of DHODH using RNA interference nor metabolic supplementation with orotate or uridine to circumvent the consequences of a loss of DHODH activity rescued the effects of DHODH inhibitors, suggesting that the effects of these inhibitors in preventing ER membrane reorganisation is most likely independent of their ability to antagonise DHODH activity. Our results strengthen the hypothesis that ER is fundamental for key mitochondrial functions, such as fusion-fission dynamics and apoptosis.

DRP-1 functions independently of mitochondrial structural perturbations to facilitate BH3 mimetic-mediated apoptosis.

Maintenance of mitochondrial integrity is critical for normal cellular homoeostasis. Most cells respond to stress stimuli and undergo apoptosis by perturbing mitochondrial structure and function to release proteins, such as cytochrome c, which are essential for the execution of the intrinsic apoptotic cascade. Cancer cells evade these events by overexpressing the anti-apoptotic BCL-2 family of proteins on mitochondrial membranes. Inhibitors of the anti-apoptotic BCL-2 family proteins, also known as BH3 mimetics, antagonise the pro-survival functions of these proteins and result in rapid apoptosis. Although the precise mechanism by which BH3 mimetics induce apoptosis has been well characterised, not much is known in terms of the structural changes that occur in mitochondria during apoptosis. Using a panel of highly selective BH3 mimetics and a wide range of cell lines, we demonstrate that BH3 mimetics induce extensive mitochondrial fission, accompanied by swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane. These changes occur in a BAX/ BAK-dependent manner. Although a major mitochondrial fission GTPase, DRP-1, has been implicated in mitochondrial apoptosis, our data demonstrate that DRP-1 might function independently/downstream of BH3 mimetic-mediated mitochondrial fission to facilitate the release of cytochrome c and apoptosis. Moreover, downregulation of DRP-1 prevented cytochrome c release and apoptosis even when OPA1, a protein mediating mitochondrial fusion, was silenced. Although BH3 mimetic-mediated displacement of BAK and other BH3-only proteins from BCL-XL and MCL-1 was unaffected by DRP-1 downregulation, it prevented BAK activation significantly, thus placing DRP-1 as one of the most critical players, along with BAX and BAK, that governs BH3 mimetic-mediated cytochrome c release and apoptosis.

BH3-only proteins are dispensable for apoptosis induced by pharmacological inhibition of both MCL-1 and BCL-XL.

The impressive selectivity and efficacy of BH3 mimetics for treating cancer has largely been limited to BCL-2 dependent hematological malignancies. Most solid tumors depend on other anti-apoptotic proteins, including MCL-1, for survival. The recent description of S63845 as the first specific and potent MCL-1 inhibitor represents an important therapeutic advance, since MCL-1 is not targeted by the currently available BH3 mimetics, Navitoclax or Venetoclax, and is commonly associated with chemoresistance. In this study, we confirm a high binding affinity and selectivity of S63845 to induce apoptosis in MCL-1-dependent cancer cell lines. Furthermore, S63845 synergizes with other BH3 mimetics to induce apoptosis in cell lines derived from both hematological and solid tumors. Although the anti-apoptotic BCL-2 family members in these cell lines interact with a spectrum of pro-apoptotic BH3-only proteins to regulate apoptosis, these interactions alone do not explain the relative sensitivities of these cell lines to BH3 mimetic-induced apoptosis. These findings necessitated further investigation into the requirement of BH3-only proteins in BH3 mimetic-mediated apoptosis. Concurrent inhibition of BCL-XL and MCL-1 by BH3 mimetics in colorectal HCT116 cells induced apoptosis in a BAX- but not BAK-dependent manner. Remarkably this apoptosis was independent of all known BH3-only proteins. Although BH3-only proteins were required for apoptosis induced as a result of BCL-XL inhibition, this requirement was overcome when both BCL-XL and MCL-1 were inhibited, implicating distinct mechanisms by which different anti-apoptotic BCL-2 family members may regulate apoptosis in cancer.

Exploring the potential of BH3 mimetic therapy in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common cancer worldwide, with overall survival of less than 50%. Current therapeutic strategies involving a combination of surgery, radiation, and/or chemotherapy are associated with debilitating side effects, highlighting the need for more specific and efficacious therapies. Inhibitors of BCL-2 family proteins (BH3 mimetics) are under investigation or in clinical practice for several hematological malignancies and show promise in solid tumors. In order to explore the therapeutic potential of BH3 mimetics in the treatment of SCCHN, we assessed the expression levels of BCL-2, BCL-XL, and MCL-1 via Western blots and immunohistochemistry, in cell lines, primary cells derived from SCCHN patients and in tissue microarrays containing tumor tissue from a cohort of 191 SCCHN patients. All preclinical models exhibited moderate to high levels of BCL-XL and MCL-1, with little or no BCL-2. Although expression levels of BCL-XL and MCL-1 did not correlate with patient outcome, a combination of BH3 mimetics to target these proteins resulted in decreased clonogenic potential and enhanced apoptosis in all preclinical models, including tumor tissue resected from patients, as well as a reduction of tumor volume in a zebrafish xenograft model of SCCHN. Our results show that SCCHN is dependent on both BCL-XL and MCL-1 for apoptosis evasion and combination therapy targeting both proteins may offer significant therapeutic benefits in this disease.

Increased hepatotoxicity of tumor necrosis factor-related apoptosis-inducing ligand in diseased human liver.

UNLABELLED: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells but not in most normal cells and has therefore been proposed as a promising antitumor agent. Recent experiments suggested that isolated primary human hepatocytes but not monkey liver cells are susceptible to certain TRAIL agonists, raising concerns about the use of TRAIL in cancer treatment. Whether TRAIL indeed exerts hepatotoxicity in vivo and how this is influenced by chemotherapeutic drugs or liver disease are completely unknown. Employing different forms of recombinant TRAIL, we found that the cytokine can induce proapoptotic caspase activity in isolated human hepatocytes. However in marked contrast, these different TRAIL preparations induced little or no cytotoxicity when incubated with tissue explants of fresh healthy liver, an experimental model that may more faithfully mimic the in vivo situation. In healthy liver, TRAIL induced apoptosis only when combined with histone deacetylase inhibitors. Strikingly, however, TRAIL alone triggered massive apoptosis accompanied by caspase activation in tissue explants from patients with liver steatosis or hepatitis C viral infection. This enhanced sensitivity of diseased liver was associated with an increased expression of TRAIL receptors and up-regulation of proapoptotic Bcl-2 proteins. CONCLUSION: These results suggest that clinical trials should be performed with great caution when TRAIL is combined with chemotherapy or administered to patients with inflammatory liver diseases.