Mitotic catastrophe can be defined as a cell death mode that occurs during or shortly after a prolonged/aberrant mitosis, and can show apoptotic or necrotic features. However, conventional procedures for the detection of apoptosis or necrosis, including biochemical bulk assays and cytofluorometric techniques, cannot discriminate among pre-mitotic, mitotic and post-mitotic death, and hence are inappropriate to monitor mitotic catastrophe. To address this issue, we generated isogenic human colon carcinoma cell lines that differ in ploidy and p53 status, yet express similar amounts of fluorescent biosensors that allow for the visualization of chromatin (histone H2B coupled to green fluorescent protein (GFP)) and centrosomes (centrin coupled to the Discosoma striata red fluorescent protein (DsRed)). By combining high-resolution fluorescence videomicroscopy and automated image analysis, we established protocols and settings for the simultaneous assessment of ploidy, mitosis, centrosome number and cell death (which in our model system occurs mainly by apoptosis). Time-lapse videomicroscopy showed that this approach can be used for the high-throughput detection of mitotic catastrophe induced by three mechanistically distinct anti-mitotic agents (dimethylenastron (DIMEN), nocodazole (NDZ) and paclitaxel (PTX)), and - in this context - revealed an important role of p53 in the control of centrosome number.

Radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using the Bcl-2/Bcl-XL inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 caused SCLC cells to acquire hallmarks of apoptosis. S44563 markedly enhanced the sensitivity of SCLC cells to radiation, as determined by a clonogenic assay. The combination of S44563 and cisplatin-based chemo-radiation showed a significant tumor growth delay and increased overall survival in mouse xenograft models. This positive interaction was greater when S44563 was given after the completion of the radiation, which might be explained by the radiation-induced overexpression of anti-apoptotic proteins secondary to activation of the NF-κB pathway. These data underline the possibility of combining IR and Bcl-2/Bcl-XL inhibition in the treatment of SCLC as they underscore the importance of administering conventional and targeted therapies in an optimal sequence.

Tumor suppressor gene p53 is a critical regulator of the cellular response to DNA damage. To examine the function of p53 in postmitotic CNS neurons, we cultured cerebellar granule cells from 15-day-old wild type and p53-deficient mice, and analyzed changes of protein expression in apoptosis elicited by DNA damage. When cerebellar granule cells from wild type mice were treated with bleomycin, a DNA strand-break inducing agent, neuronal death occurred. In contrast, cells from p53-deficient mice were resistant to bleomycin-induced neuronal death. Furthermore, cells from p53 heterozygous mice showed an intermediate resistance between wild type and p53-deficient mice. These results show that p53 is required for the bleomycin-induced cerebellar granule cell death. To examine which proteins are involved in this apoptosis, we examined changes in protein levels of the Bcl-2 family, including Bcl-2, Bcl-X and Bax. The relative amounts of these proteins did not change after bleomycin treatment, suggesting that the changes in the levels of these Bcl-2 family proteins are not necessary for apoptosis in this system. In contrast, the levels of c-Jun protein significantly increased 6 h after treatment with bleomycin in wild type but not in p53-deficient cerebellar granule cells. These results raise the possibility that c-Jun is required for p53-dependent neuronal apoptosis induced by bleomycin.

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.

The BAG family of Hsp70/Hsc70 co-chaperones is characterised by the presence of a conserved BAG domain at the carboxyl-terminus. BAG3 protein is the only member of this family containing also the N-terminally located WW domain. We describe here the identification of adenovirus (Ad) penton base protein as the first BAG3 partner recognising BAG3 WW domain. Ad penton base is the viral capsid constituent responsible for virus internalisation. It contains in the N-terminal part two conserved PPxY motifs, known ligands of WW domains. In cells producing Ad penton base protein, cytoplasmic endogenous BAG3 interacts with it and co-migrates to the nucleus. Preincubation of BAG3 with Ad base protein results in only slight modulation of BAG3 co-chaperone activity, suggesting that this interaction is not related to the classical BAG3 co-chaperone function. However, depletion of BAG3 impairs the cell entry of the virus and viral progeny production in Ad-infected cells, suggesting that the interaction between virus penton base protein and cellular co-chaperone BAG3 positively influences virus life cycle. These results thus demonstrate a novel host-pathogen interaction, which contributes to the successful infectious life cycle of adenoviruses. In addition, these data enrich our knowledge about the multifunctionality of the BAG3 co-chaperone.

Chronic pancreatitis (CP) is associated with elevated plasma levels of bacterial lipopolysaccharide (LPS) and we have demonstrated reduced acinar cell autophagy in human CP tissue. Therefore, we investigated the role of autophagy in experimental endotoxin-induced pancreatic injury and aimed to identify LPS in human CP tissue. Pancreatic Atg7-deficient mice were injected with a single sub-lethal dose of LPS. Expression of autophagy, apoptosis, necroptosis, and inflammatory markers was determined 3 and 24 h later utilizing immunoblotting and immunofluorescence. The presence of LPS in pancreatic tissue from mice and from patients and healthy controls was determined using immunohistochemistry, immunoblots, and chromogenic assay. Mice lacking pancreatic autophagy exhibited local signs of inflammation and were particularly sensitive to the toxic effect of LPS injection as compared to control mice. In response to LPS, Atg7Δpan mice exhibited enhanced vacuolization of pancreatic acinar cells, increase in TLR4 expression coupled to enhanced expression of NF-κΒ, JNK, and pro-inflammatory cytokines by acinar cells and enhanced infiltration by myeloid cells (but not Atg7F/F controls). Cell death was enhanced in Atg7Δpan pancreata, but only necroptosis and trypsin activation was further amplified following LPS injection along with elevated pancreatic LPS. The presence of LPS was identified in the pancreata from all 14 CP patients examined but was absent in the pancreata from all 10 normal controls. Altogether, these results support a potential role for metabolic endotoxemia in the pathogenesis of CP. Moreover, the evidence also supports the notion that autophagy plays a major cytoprotective and anti-inflammatory role in the pancreas, and blunting metabolic endotoxemia-induced CP.

Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Apaf-1(-/-) or caspase-3(-/-) cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1(-/-) or caspase-3(-/-) cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1(-/-) or caspase-3(-/-) cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

Cell death in B cell terminal differentiation rapidly follows cell cycle arrest in IL-6 differentiation of EBV-immortalized, IgG-bearing human lymphoblastoid cells in vitro. G1 arrest is now found to coincide with repression of EBNA2 and LMP1, two EBV genes essential for B cell transformation, without activation of the viral lytic cycle. IL-6-differentiated B cells die by apoptosis, as evidenced by increases in Annexin V binding activity, PARP cleavage, and chromatin disorganization. Expression of Mcl-1, a Bcl-2 family member, was specifically induced during IL-6 differentiation and down-regulated during apoptosis. Thus, IL-6 reverses EBV immortalization and activates the terminal differentiation program in IgG-bearing human B lymphoblastoid cells, including regulation of an anti-apoptotic gene to coordinate differentiation, cell cycle arrest, and cell death.

According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.

The mammalian proapoptotic protein Bax confers a lethal phenotype when expressed in yeast. By exploiting this phenotype, we have identified a novel human Bax inhibitor, BI-1. BI-1 is an evolutionarily conserved integral membrane protein containing multiple membrane-spanning segments and is predominantly localized to intracellular membranes, similar to Bcl-2 family proteins. Moreover, BI-1 can interact with Bcl-2 and Bcl-XL but Bax or Bak, as demonstrated by in vivo cross-linking and coimmunoprecipitation studies. When overexpressed in mammalian cells, BI-1 suppressed apoptosis included by Bax, etoposide, staurosporine, and growth factor deprivation, but not by Fas (CD95). Conversely, BI-1 antisense induced apoptosis. BI-1 thus represents a new type of regulator of cell death pathways controlled by Bcl-2 and Bax.

The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.

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.

Platinum-based drugs remain as the cornerstone of cancer chemotherapy; however, development of multidrug resistance presents a therapeutic challenge. This study aims at understanding the molecular mechanisms underlying resistance to cisplatin and unraveling surrogate signaling networks that could revert sensitivity to apoptosis stimuli. We made use of three different sets of cell lines, A549 and H2030 non-small-cell lung cancer (NSCLC) and A2780 ovarian cancer cells and their cisplatin-resistant variants. Here we report that cisplatin-resistant cell lines displayed a multidrug-resistant phenotype. Changes in mitochondrial metabolism and defective mitochondrial signaling were unraveled in the resistant cells. More interestingly, a marked increase in sensitivity of the resistant cells to death receptor-induced apoptosis, in particular TRAIL (TNF-related apoptosis-inducing ligand)-mediated execution, was observed. Although this was not associated with an increase in gene transcription, a significant increase in the localization of TRAIL death receptor, DR4, to the lipid raft subdomains of plasma membrane was detected in the resistant variants. Furthermore, exposure of cisplatin-resistant cells to TRAIL resulted in upregulation of inducible nitric oxide synthase (iNOS) and increase in nitric oxide (NO) production that triggered the generation of peroxynitrite (ONOO(-)). Scavenging ONOO(-) rescued cells from TRAIL-induced apoptosis, thereby suggesting a critical role of ONOO(-) in TRAIL-induced execution of cisplatin-resistant cells. Notably, preincubation of cells with TRAIL restored sensitivity of resistant cells to cisplatin. These data provide compelling evidence for employing strategies to trigger death receptor signaling as a second-line treatment for cisplatin-resistant cancers.

LTX-315 is a cationic amphilytic peptide that preferentially permeabilizes mitochondrial membranes, thereby causing partially BAX/BAK1-regulated, caspase-independent necrosis. Based on the observation that intratumorally injected LTX-315 stimulates a strong T lymphocyte-mediated anticancer immune response, we investigated whether LTX-315 may elicit the hallmarks of immunogenic cell death (ICD), namely (i) exposure of calreticulin on the plasma membrane surface, (ii) release of ATP into the extracellular space, (iii) exodus of HMGB1 from the nucleus, and (iv) induction of a type-1 interferon response. Using a panel of biosensor cell lines and robotized fluorescence microscopy coupled to automatic image analysis, we observed that LTX-315 induces all known ICD characteristics. This conclusion was validated by several independent methods including immunofluorescence stainings (for calreticulin), bioluminescence assays (for ATP), immunoassays (for HMGB1), and RT-PCRs (for type-1 interferon induction). When injected into established cancers, LTX-315 caused a transiently hemorrhagic focal necrosis that was accompanied by massive release of HMGB1 (from close-to-all cancer cells), as well as caspase-3 activation in a fraction of the cells. LTX-315 was at least as efficient as the positive control, the anthracycline mitoxantrone (MTX), in inducing local inflammation with infiltration by myeloid cells and T lymphocytes. Collectively, these results support the idea that LTX-315 can induce ICD, hence explaining its capacity to mediate immune-dependent therapeutic effects.

Clinical oncology heavily relies on the use of radiotherapy, which often leads to merely transient responses that are followed by local or distant relapse. The molecular mechanisms explaining radioresistance are largely elusive. Here, we identified a dual role of autophagy in the response of cancer cells to ionizing radiation. On one hand, we observed that the depletion of essential autophagy-relevant gene products, such as ATG5 and Beclin 1, increased the sensitivity of human or mouse cancer cell lines to irradiation, both in vitro (where autophagy inhibition increased radiation-induced cell death and decreased clonogenic survival) and in vivo, after transplantation of the cell lines into immunodeficient mice (where autophagy inhibition potentiated the tumour growth-inhibitory effect of radiotherapy). On the other hand, when tumour proficient or deficient for autophagy were implanted in immunocompetent mice, it turned out that defective autophagy reduced the efficacy of radiotherapy. Indeed, radiotherapy elicited an anti-cancer immune response that was dependent on autophagy-induced ATP release from stressed or dying tumour cells and was characterized by dense lymphocyte infiltration of the tumour bed. Intratumoural injection of an ecto-ATPase inhibitor restored the immune infiltration of autophagy-deficient tumours post radiotherapy and improved the growth-inhibitory effect of ionizing irradiation. Altogether, our results reveal that beyond its cytoprotective function, autophagy confers immunogenic properties to tumours, hence amplifying the efficacy of radiotherapy in an immunocompetent context. This has far-reaching implications for the development of pharmacological radiosensitizers.

Tumor necrosis factor-α (TNF-α) has important roles in several immunological events by regulating apoptosis and transcriptional activation of cytokine genes. Intracellular signaling mediated by TNF-receptor-type 1 (TNFR1) is constituted by two sequential protein complexes: Complex-I containing the receptor and Complex-II-containing Caspase-8. Protein modifications, particularly ubiquitination, are associated with the regulation of the formation of these complexes. However, the underlying mechanisms remain poorly defined. Here, we identified CLIP-170-related 59 kDa protein (CLIPR-59) as a novel adaptor protein for TNFR1. Experimental reduction of CLIPR-59 levels prevented induction of apoptosis and activation of caspases in the context of TNF-α signaling. CLIPR-59 binds TNFR1 but dissociates in response to TNF-α stimulation. However, CLIPR-59 is also involved in and needed for the formation of Complex-II. Moreover, CLIPR-59 regulates TNF-α-induced ubiquitination of receptor-interacting protein 1 (RIP1) by its association with CYLD, a de-ubiquitinating enzyme. These findings suggest that CLIPR-59 modulates ubiquitination of RIP1, resulting in the formation of Complex-II and thus promoting Caspase-8 activation to induce apoptosis by TNF-α.

Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.