Simone Fulda

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. One of the most important advances in cancer research in recent years is the recognition that cell death mostly by apoptosis is crucially involved in the regulation of tumor formation and also critically determines treatment response. Killing of tumor cells by most anticancer strategies currently used in clinical oncology, for example, chemotherapy, gamma-irradiation, suicide gene therapy or immunotherapy, has been linked to activation of apoptosis signal transduction pathways in cancer cells such as the intrinsic and/or extrinsic pathway. Thus, failure to undergo apoptosis may result in treatment resistance. Understanding the molecular events that regulate apoptosis in response to anticancer chemotherapy, and how cancer cells evade apoptotic death, provides novel opportunities for a more rational approach to develop molecular-targeted therapies for combating cancer.

Mitochondria are the cells' powerhouse, but also their suicidal weapon store. Dozens of lethal signal transduction pathways converge on mitochondria to cause the permeabilization of the mitochondrial outer membrane, leading to the cytosolic release of pro-apoptotic proteins and to the impairment of the bioenergetic functions of mitochondria. The mitochondrial metabolism of cancer cells is deregulated owing to the use of glycolytic intermediates, which are normally destined for oxidative phosphorylation, in anabolic reactions. Activation of the cell death machinery in cancer cells by inhibiting tumour-specific alterations of the mitochondrial metabolism or by stimulating mitochondrial membrane permeabilization could therefore be promising therapeutic approaches.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

Pan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.

Cells can respond to stress in various ways ranging from the activation of survival pathways to the initiation of cell death that eventually eliminates damaged cells. Whether cells mount a protective or destructive stress response depends to a large extent on the nature and duration of the stress as well as the cell type. Also, there is often the interplay between these responses that ultimately determines the fate of the stressed cell. The mechanism by which a cell dies (i.e., apoptosis, necrosis, pyroptosis, or autophagic cell death) depends on various exogenous factors as well as the cell's ability to handle the stress to which it is exposed. The implications of cellular stress responses to human physiology and diseases are manifold and will be discussed in this review in the context of some major world health issues such as diabetes, Parkinson's disease, myocardial infarction, and cancer.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

The endoplasmic reticulum ( ER ) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one‐third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response ( UPR ), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling‐centric view of ER functions, from the ER 's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR , its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.

One of the hallmarks of human cancers is the intrinsic or acquired resistance to apoptosis. Evasion of apoptosis may contribute to carcinogenesis, tumor progression and also to treatment resistance, since most current anticancer therapies including chemotherapy, radio- and immunotherapy primarily act by activating cell death pathways including apoptosis in cancer cells. Hence, a better understanding of the molecular mechanisms underlying tumor resistance to apoptotic cell death is expected to provide the basis for a rational approach to develop molecular targeted therapies.

High-risk neuroblastoma is a devastating malignancy with very limited therapeutic options. Here, we identify withaferin A (WA) as a natural ferroptosis-inducing agent in neuroblastoma, which acts through a novel double-edged mechanism. WA dose-dependently either activates the nuclear factor-like 2 pathway through targeting of Kelch-like ECH-associated protein 1 (noncanonical ferroptosis induction) or inactivates glutathione peroxidase 4 (canonical ferroptosis induction). Noncanonical ferroptosis induction is characterized by an increase in intracellular labile Fe(II) upon excessive activation of heme oxygenase-1, which is sufficient to induce ferroptosis. This double-edged mechanism might explain the superior efficacy of WA as compared with etoposide or cisplatin in killing a heterogeneous panel of high-risk neuroblastoma cells, and in suppressing the growth and relapse rate of neuroblastoma xenografts. Nano-targeting of WA allows systemic application and suppressed tumor growth due to an enhanced accumulation at the tumor site. Collectively, our data propose a novel therapeutic strategy to efficiently kill cancer cells by ferroptosis.

Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFalpha, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2'-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.

Primary or acquired resistance to current treatment protocols remains a major concern in clinical oncology and may be caused by defects in apoptosis programs. Since recent data suggest that TRAIL can bypass apoptosis resistance caused by Bcl-2, we further investigated the role of Bcl-2 in TRAIL-induced apoptosis. Here we report that overexpression of Bcl-2 conferred protection against TRAIL in neuroblastoma, glioblastoma or breast carcinoma cell lines. Bcl-2 overexpression reduced TRAIL-induced cleavage of caspase-8 and Bid indicating that caspase-8 was activated upstream and also downstream of mitochondria in a feedback amplification loop. Importantly, Bcl-2 blocked cleavage of caspases-9, -7 and -3 into active subunits and cleavage of the caspase substrates DFF45 or PARP. Also, Bcl-2 blocked cleavage of XIAP and overexpression of XIAP conferred resistance against TRAIL indicating that apoptosis was also amplified through a feedforward loop between caspases and XIAP. In contrast, in SKW lymphoblastoid cells, TRAIL-induced activation of caspase-8 directly translated into full activation of caspases, cleavage of XIAP, DFF45 or PARP and apoptosis independent of Bcl-2 overexpression, although Bcl-2 similarly inhibited loss of mitochondrial membrane potential and the release of cytochrome c, AIF and Smac from mitochondria in all cell types. By demonstrating a cell type dependent regulation of the TRAIL signaling pathway at different level, e.g. by Bcl-2 and by XIAP, these findings may have important clinical implication. Thus, strategies targeting the molecular basis of resistance towards TRAIL may be necessary in some tumors for cancer therapy with TRAIL.

Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochromec to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria. Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochromec to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria. permeability transition apoptosis-inducing factor betulinic acid fluorescence-activated cell sorting benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone bongkrekic acid phosphate-buffered saline polyacrylamide gel electrophoresis bovine serum albumin poly(ADP-ribose) polymerase monoclonal antibody 1,4-piperazinediethanesulfonic acid mitochondrial transmembrane potential. Anticancer agents with different modes of action have been reported to trigger apoptosis in chemosensitive cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Alterations of mitochondrial functions such as permeability transition (PT)1 have been found to play a major role in the apoptotic process including cell death induced by chemotherapeutic agents (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 4Marchetti P. Castedo M. Susin S.A. Zamzami N. Hirsch T. Macho A. Haeffner A. Hirsch T. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1155-1160Crossref PubMed Scopus (779) Google Scholar, 5Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. 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These findings suggest that caspases can act upstream and downstream of mitochondria. Mitochondrial function during apoptosis is controlled by the Bcl-2 family of proteins localized to intracellular membranes including the mitochondrial membrane (13Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1706) Google Scholar). Overexpression of the anti-apoptotic molecules Bcl-2 and Bcl-XL has been found to confer resistance to anticancer treatment (14Kim C.N. Wang X. Huang Y. Ibrado A.M. Liu L. Fang G. Bhalla K. Cancer Res. 1997; 57: 3115-3120PubMed Google Scholar, 15Dole M. Nuñez G. Merchant A.K. Maybaum J. Rode C.K. Bloch C.A. Castle V.P. Cancer Res. 1994; 54: 3253-3259PubMed Google Scholar, 16Dole M.G. Jasty R. Cooper M.J. Thompson C.B. Nuñez G. Castle V.P. Cancer Res. 1995; 55: 2576-2582PubMed Google Scholar). Bcl-2 and Bcl-XL may inhibit apoptosis through the capacity to prevent PT and/or to stabilize the barrier function of the outer mitochondrial membrane (5Zamzami N. Susin S.A. Marchetti P. Hirsch T. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 13Kroemer G. Nat. Med. 1997; 3: 614-620Crossref PubMed Scopus (1706) Google Scholar, 17Vander Heiden M.G. Chandel N.S. Williamson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1233) Google Scholar, 18Bossy-Wetzel E. Newmeyer D.D. Green D.R. EMBO J. 1998; 17: 37-49Crossref PubMed Scopus (1105) Google Scholar).Cytotoxic drugs such as doxorubicin can activate apoptosis pathways by inducing ligand/receptor-driven amplifier systems such as the CD95 system (19Friesen C. Herr I. Krammer P.H. Debatin K.-M. Nat. Med. 1996; 2: 574-577Crossref PubMed Scopus (951) Google Scholar, 20Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.-M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar, 21Fulda S. Los M. Friesen C. Debatin K.-M. Int. J. Cancer. 1998; 76: 105-114Crossref PubMed Scopus (165) Google Scholar, 22Fulda S. Scaffidi C. Pietsch T. Krammer P.H. Peter M.E. Debatin K.-M. Cell Death Differ. 1998; 5: 884-893Crossref PubMed Scopus (117) Google Scholar, 23Los M. Herr I. Friesen C. Fulda S. Schulze-Osthoff K. Debatin K.-M. Blood. 1997; 90: 3118-3129Crossref PubMed Google Scholar, 24Friesen C. Fulda S. Debatin K.-M. Leukemia (Baltimore). 1997; 1: 1833-1841Crossref Scopus (189) Google Scholar, 25Debatin K.-M. J. Natl. Cancer Inst. 1997; 89: 750-751Crossref PubMed Scopus (61) Google Scholar, 26Mueller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (715) Google Scholar). Upon CD95 ligand/receptor interaction, caspase-8 (FLICE/MACH/Mch5) is cleaved, resulting in activation of a downstream caspase cascade including caspase-3 (CPP32/Yama) (27Oehm A. Behrmann I. Falk W. Pawlita M. Maier G. Klas C. Li-Weber M. Richards S. Dhein J. Trauth B.C. Ponstingl H. Krammer P.H. J. Biol. Chem. 1992; 267: 10709-10715Abstract Full Text PDF PubMed Google Scholar, 28Trauth B.C. Klas C. Peter A.M.J. Matz S. Möller P. Falk W. Debatin K.-M. Krammer P.H. Science. 1989; 245: 301-305Crossref PubMed Scopus (1655) Google Scholar, 29Debatin K.-M. Cell Death Differ. 1996; 3: 185-189PubMed Google Scholar, 30Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4537) Google Scholar, 31Peter M.E. Kischkel F.C. Hellbrandt S. Chinnaiyan A.M. Krammer P.H. Dixit V.M. Cell Death Differ. 1996; 3: 161-170PubMed Google Scholar, 32Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A.N.J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2723) Google Scholar, 33Chinnaiyan A.M. Teppert C.G. Seldin M.F. O'Rourke K. Kischkel F.C. Hellbardt S. Krammer P.H. Peter M.E. Dixit V.M. J. Biol. Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 34Kischkel F.C. Hellbrandt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1770) Google Scholar, 35Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A.N.J. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1038) Google Scholar). Induction of the CD95 ligand and up-regulation of CD95 after treatment with cytotoxic drugs such as doxorubicin have been observed in a variety of tumor cells, and blockade of CD95/CD95 ligand interaction by antagonistic antibodies has been found to inhibit drug-induced cell death (19Friesen C. Herr I. Krammer P.H. Debatin K.-M. Nat. Med. 1996; 2: 574-577Crossref PubMed Scopus (951) Google Scholar, 20Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.-M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar, 21Fulda S. Los M. Friesen C. Debatin K.-M. Int. J. Cancer. 1998; 76: 105-114Crossref PubMed Scopus (165) Google Scholar, 22Fulda S. Scaffidi C. Pietsch T. Krammer P.H. Peter M.E. Debatin K.-M. Cell Death Differ. 1998; 5: 884-893Crossref PubMed Scopus (117) Google Scholar, 23Los M. Herr I. Friesen C. Fulda S. Schulze-Osthoff K. Debatin K.-M. Blood. 1997; 90: 3118-3129Crossref PubMed Google Scholar, 24Friesen C. Fulda S. Debatin K.-M. Leukemia (Baltimore). 1997; 1: 1833-1841Crossref Scopus (189) Google Scholar, 25Debatin K.-M. J. Natl. Cancer Inst. 1997; 89: 750-751Crossref PubMed Scopus (61) Google Scholar, 26Mueller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (715) Google Scholar).Betulinic acid (BetA) is a novel anticancer drug with specificity for neuroectodermal tumors (36Pisha E. Chai H. Lee I.S Chagwedera T.E. Farnsworth N.R. Cordell G.A. Beecher C.W. Fong H.H. Kinghorn A.D. Brown D.M. Wani M.C. Wall M.E. Hieken T.J. Das Gupta T.K. Pezzuto J.M. Nat. Med. 1995; 1: 1046-1051Crossref PubMed Scopus (773) Google Scholar, 37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). We previously found that BetA-induced apoptosis differs from “classical” anticancer agents such as doxorubicin (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). BetA-induced apoptosis is not associated with activation of ligand/receptor systems such as CD95 and does not involve p53. Perturbation of mitochondrial function including loss of mitochondrial permeability transition precedes other key features of apoptosis such as activation of the caspase cascade and nuclear fragmentation (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). This suggests that BetA may have a direct effect on mitochondria. We therefore asked whether BetA would directly activate mitochondria and studied the sequence of the BetA-triggered apoptosis pathway.DISCUSSIONCytotoxic drugs have been reported to act primarily by inducing apoptosis in sensitive target cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Triggering of apoptosis by anticancer drugs involves simultaneous or subsequent activation of death receptor systems, perturbation of mitochondrial function, and proteolytic processing of caspases, the death effector molecules of apoptosis (25Debatin K.-M. J. Natl. Cancer Inst. 1997; 89: 750-751Crossref PubMed Scopus (61) Google Scholar). Thus, the cell death pathway may be entered at multiple sites, and most drugs may hit various targets, although the precise molecular mechanisms have not been characterized in detail. Here, we report that one class of anticancer agents exemplified by BetA may act by directly targeting mitochondria, resulting in caspase activation downstream of mitochondria.Using a cell-free system, we found that BetA directly triggered PT in isolated mitochondria, and induction of PT appears to be the initial event in BetA-triggered apoptosis. Inhibition of PT by overexpression of Bcl-2 or Bcl-XL or by the mitochondrion-specific inhibitor BA prevented all manifestations of apoptosis in intact cells and in a cell-free system such as disruption of ΔΨm, activation of caspases, cleavage of substrates (PARP), and nuclear fragmentation. In contrast to BetA, classical cytotoxic drugs such as doxorubicin, cis-platinum, or etoposide did not induce mitochondrial perturbations in isolated mitochondria, suggesting that mitochondrial PT, which occurred in intact cells during apoptosis triggered by these substances (20Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.-M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar), was the consequence of a primary activation of other pathways or systems. BetA specifically kills neuroectodermal tumor cells (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). When added to intact cells, BetA specifically induced mitochondrial alterations in SHEP neuroblastoma cells, but not in lymphoid cell lines (data not shown). However, BetA-treated mitochondria isolated from the B lymphoblastoid cell line SKW6.4 triggered mitochondrial PT and mediated nuclear fragmentation, similar to mitochondria from SHEP cells. Thus, the specificity of BetA for neuroectodermal tumors may be explained by cell-specific uptake and/or translocation of the compound to the mitochondrial compartment rather than by differences in mitochondria themselves.In BetA-treated neuroblastoma cells, activation of different caspases and nuclear fragmentation were found only in cells with perturbed mitochondrial function, and BetA-induced loss of ΔΨm could not be inhibited by the caspase inhibitor Z-VAD-fmk. This suggests that caspase activation occurs downstream of mitochondria and that activation of mitochondria is sufficient to trigger all downstream events leading to apoptosis. In contrast, in doxorubicin-treated cells, loss of ΔΨm was inhibited by Z-VAD-fmk, indicating that activation of mitochondria is preceded by upstream caspase activation. 2Fulda, S., Susin, S. A., Kroemer, G., and Debatin, K.-M. (1998) Cancer Res. 58,4453–4460. Thus, BetA seems to define a class of cytotoxic agents whose apoptosis-inducing effect is predominately initiated by activation of mitochondria.Our studies on BetA-induced apoptosis provide a molecular sequence of the interplay between activation of different caspases and mitochondrial PT. Following death receptor triggering, the receptor proximal caspase-8 becomes activated, which in turn mediates full activation of the caspase cascade, cleavage of substrates, and concomitant triggering of mitochondrial PT (40Scaffidi C. Fulda S. Li F. Friesen C. Srinivasan A. Tomaselli K.J. Debatin K.-M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2617) Google Scholar, 41Medema J.P. Scaffidi C. Krammer P.H. Peter M.E. J. Biol. Chem. 1998; 273: 3388-3393Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 42Srinivasan A. Li F. Wong A. Kodandapani L. Smidt Jr., R. Krebs J.F. Fritz L.C. Wu J.C. Tomaselli K.J J. Biol. Chem. 1998; 273: 4523-4529Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 43Boise L.H. Thompson C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3759-3764Crossref PubMed Scopus (207) Google Scholar). Therefore, upon CD95 triggering, activation of caspase-8 occurs even in cells in which mitochondrial PT and processing of downstream caspases are blocked by overexpression of Bcl-2 or Bcl-XL (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). In contrast, in BetA-treated cells, activation of both caspase-8 and caspase-3 was secondary to mitochondrial PT. Apoptogenic proteins such as cytochromec and AIF released from mitochondria upon permeability transition have been shown to directly induce cleavage of caspases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar,3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar). BetA-triggered mitochondrial PT and apoptosis involved cleavage of caspase-8 and caspase-3 independent of CD95 ligand/receptor interaction. Thus, in BetA-treated cells, caspase-8 became activated by mitochondria undergoing PT in the absence of CD95 death-inducing signaling complex formation. AIF released from mitochondria may mediate caspase-8 cleavage following BetA treatment since partially purified AIF could cleave recombinant caspase-8. In contrast, cytochromec did not induce activation of caspase-8, although it induced caspase-3 cleavage, indicating that the mechanism of caspase-8 activation downstream of mitochondria differed from that of caspase-3. Caspases might appear to be dispensable for mediating DNA fragmentation since partially purified AIF has been described to induce DNA fragmentation and since we found fragmentation of nuclei upon incubation with isolated mitochondria. However, these preparations may still contain small amounts of cytosolic fractions that may mediate activation of downstream targets such as PARP, DNA fragmentation factor, or caspase-activated deoxyribonuclease (44Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 45Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1997; 391: 43-50Crossref Scopus (2796) Google Scholar).Different classes of anticancer drugs may enter the apoptotic pathway at distinct entry sites before they eventually induce a Bcl-2/Bcl-XL-controlled ΔΨm disruption that marks the initiation of a common effector phase of apoptosis. Our findings may have implications for tumor therapy directed toward activation of apoptosis effector systems. Direct activation of mitochondrial PT, as exemplified by cytotoxic drugs such as BetA, may be sufficient for induction of apoptosis in cancer cells and may bypass the requirement for upstream signaling. Those drugs could still be effective against tumor cells that have a defect in upstream apoptosis pathways. In this context, it appears intriguing that tumors cells with a defect in the CD95 system (24Friesen C. Fulda S. Debatin K.-M. Leukemia (Baltimore). 1997; 1: 1833-1841Crossref Scopus (189) Google Scholar), which fail to respond to classical chemotherapeutic agents, are fully susceptible to BetA-induced cell death (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). Thus, our findings may be important to define and develop a new class of cytotoxic agents with direct mitochondrial effects that could overcome some forms of tumor-associated mechanisms of chemoresistance. Anticancer agents with different modes of action have been reported to trigger apoptosis in chemosensitive cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Alterations of mitochondrial functions such as permeability transition (PT)1 have been found to play a major role in the apoptotic process including cell death induced by chemotherapeutic agents (2Kroemer G. Zamzami N. Susin S.A. 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Mitochondria undergoing PT release apoptogenic proteins such as cytochrome c or apoptosis-inducing factor (AIF) from the mitochondrial intermembrane space into the cytosol, where they can activate caspases and endonucleases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar, 3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 8Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4256) Google Scholar, 9Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4384) Google Scholar, 10Liu X. Kim C.N. Yang J. 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Debatin K.-M. Cell Death Differ. 1998; 5: 884-893Crossref PubMed Scopus (117) Google Scholar, 23Los M. Herr I. Friesen C. Fulda S. Schulze-Osthoff K. Debatin K.-M. Blood. 1997; 90: 3118-3129Crossref PubMed Google Scholar, 24Friesen C. Fulda S. Debatin K.-M. Leukemia (Baltimore). 1997; 1: 1833-1841Crossref Scopus (189) Google Scholar, 25Debatin K.-M. J. Natl. Cancer Inst. 1997; 89: 750-751Crossref PubMed Scopus (61) Google Scholar, 26Mueller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (715) Google Scholar). Upon CD95 ligand/receptor interaction, caspase-8 (FLICE/MACH/Mch5) is cleaved, resulting in activation of a downstream caspase cascade including caspase-3 (CPP32/Yama) (27Oehm A. Behrmann I. Falk W. Pawlita M. Maier G. Klas C. Li-Weber M. Richards S. Dhein J. Trauth B.C. Ponstingl H. Krammer P.H. J. Biol. 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Chem. 1996; 271: 4961-4965Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 34Kischkel F.C. Hellbrandt S. Behrmann I. Germer M. Pawlita M. Krammer P.H. Peter M.E. EMBO J. 1995; 14: 5579-5588Crossref PubMed Scopus (1770) Google Scholar, 35Medema J.P. Scaffidi C. Kischkel F.C. Shevchenko A.N.J. Mann M. Krammer P.H. Peter M.E. EMBO J. 1997; 16: 2794-2804Crossref PubMed Scopus (1038) Google Scholar). Induction of the CD95 ligand and up-regulation of CD95 after treatment with cytotoxic drugs such as doxorubicin have been observed in a variety of tumor cells, and blockade of CD95/CD95 ligand interaction by antagonistic antibodies has been found to inhibit drug-induced cell death (19Friesen C. Herr I. Krammer P.H. Debatin K.-M. Nat. Med. 1996; 2: 574-577Crossref PubMed Scopus (951) Google Scholar, 20Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.-M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar, 21Fulda S. Los M. Friesen C. Debatin K.-M. Int. J. Cancer. 1998; 76: 105-114Crossref PubMed Scopus (165) Google Scholar, 22Fulda S. Scaffidi C. Pietsch T. Krammer P.H. Peter M.E. Debatin K.-M. Cell Death Differ. 1998; 5: 884-893Crossref PubMed Scopus (117) Google Scholar, 23Los M. Herr I. Friesen C. Fulda S. Schulze-Osthoff K. Debatin K.-M. Blood. 1997; 90: 3118-3129Crossref PubMed Google Scholar, 24Friesen C. Fulda S. Debatin K.-M. Leukemia (Baltimore). 1997; 1: 1833-1841Crossref Scopus (189) Google Scholar, 25Debatin K.-M. J. Natl. Cancer Inst. 1997; 89: 750-751Crossref PubMed Scopus (61) Google Scholar, 26Mueller M. Strand S. Hug H. Heinemann E.-M. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P.R. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (715) Google Scholar). Betulinic acid (BetA) is a novel anticancer drug with specificity for neuroectodermal tumors (36Pisha E. Chai H. Lee I.S Chagwedera T.E. Farnsworth N.R. Cordell G.A. Beecher C.W. Fong H.H. Kinghorn A.D. Brown D.M. Wani M.C. Wall M.E. Hieken T.J. Das Gupta T.K. Pezzuto J.M. Nat. Med. 1995; 1: 1046-1051Crossref PubMed Scopus (773) Google Scholar, 37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). We previously found that BetA-induced apoptosis differs from “classical” anticancer agents such as doxorubicin (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). BetA-induced apoptosis is not associated with activation of ligand/receptor systems such as CD95 and does not involve p53. Perturbation of mitochondrial function including loss of mitochondrial permeability transition precedes other key features of apoptosis such as activation of the caspase cascade and nuclear fragmentation (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). This suggests that BetA may have a direct effect on mitochondria. We therefore asked whether BetA would directly activate mitochondria and studied the sequence of the BetA-triggered apoptosis pathway. DISCUSSIONCytotoxic drugs have been reported to act primarily by inducing apoptosis in sensitive target cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Triggering of apoptosis by anticancer drugs involves simultaneous or subsequent activation of death receptor systems, perturbation of mitochondrial function, and proteolytic processing of caspases, the death effector molecules of apoptosis (25Debatin K.-M. J. Natl. Cancer Inst. 1997; 89: 750-751Crossref PubMed Scopus (61) Google Scholar). Thus, the cell death pathway may be entered at multiple sites, and most drugs may hit various targets, although the precise molecular mechanisms have not been characterized in detail. Here, we report that one class of anticancer agents exemplified by BetA may act by directly targeting mitochondria, resulting in caspase activation downstream of mitochondria.Using a cell-free system, we found that BetA directly triggered PT in isolated mitochondria, and induction of PT appears to be the initial event in BetA-triggered apoptosis. Inhibition of PT by overexpression of Bcl-2 or Bcl-XL or by the mitochondrion-specific inhibitor BA prevented all manifestations of apoptosis in intact cells and in a cell-free system such as disruption of ΔΨm, activation of caspases, cleavage of substrates (PARP), and nuclear fragmentation. In contrast to BetA, classical cytotoxic drugs such as doxorubicin, cis-platinum, or etoposide did not induce mitochondrial perturbations in isolated mitochondria, suggesting that mitochondrial PT, which occurred in intact cells during apoptosis triggered by these substances (20Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.-M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar), was the consequence of a primary activation of other pathways or systems. BetA specifically kills neuroectodermal tumor cells (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). When added to intact cells, BetA specifically induced mitochondrial alterations in SHEP neuroblastoma cells, but not in lymphoid cell lines (data not shown). However, BetA-treated mitochondria isolated from the B lymphoblastoid cell line SKW6.4 triggered mitochondrial PT and mediated nuclear fragmentation, similar to mitochondria from SHEP cells. Thus, the specificity of BetA for neuroectodermal tumors may be explained by cell-specific uptake and/or translocation of the compound to the mitochondrial compartment rather than by differences in mitochondria themselves.In BetA-treated neuroblastoma cells, activation of different caspases and nuclear fragmentation were found only in cells with perturbed mitochondrial function, and BetA-induced loss of ΔΨm could not be inhibited by the caspase inhibitor Z-VAD-fmk. This suggests that caspase activation occurs downstream of mitochondria and that activation of mitochondria is sufficient to trigger all downstream events leading to apoptosis. In contrast, in doxorubicin-treated cells, loss of ΔΨm was inhibited by Z-VAD-fmk, indicating that activation of mitochondria is preceded by upstream caspase activation. 2Fulda, S., Susin, S. A., Kroemer, G., and Debatin, K.-M. (1998) Cancer Res. 58,4453–4460. Thus, BetA seems to define a class of cytotoxic agents whose apoptosis-inducing effect is predominately initiated by activation of mitochondria.Our studies on BetA-induced apoptosis provide a molecular sequence of the interplay between activation of different caspases and mitochondrial PT. Following death receptor triggering, the receptor proximal caspase-8 becomes activated, which in turn mediates full activation of the caspase cascade, cleavage of substrates, and concomitant triggering of mitochondrial PT (40Scaffidi C. Fulda S. Li F. Friesen C. Srinivasan A. Tomaselli K.J. Debatin K.-M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2617) Google Scholar, 41Medema J.P. Scaffidi C. Krammer P.H. Peter M.E. J. Biol. Chem. 1998; 273: 3388-3393Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 42Srinivasan A. Li F. Wong A. Kodandapani L. Smidt Jr., R. Krebs J.F. Fritz L.C. Wu J.C. Tomaselli K.J J. Biol. Chem. 1998; 273: 4523-4529Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 43Boise L.H. Thompson C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3759-3764Crossref PubMed Scopus (207) Google Scholar). Therefore, upon CD95 triggering, activation of caspase-8 occurs even in cells in which mitochondrial PT and processing of downstream caspases are blocked by overexpression of Bcl-2 or Bcl-XL (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). In contrast, in BetA-treated cells, activation of both caspase-8 and caspase-3 was secondary to mitochondrial PT. Apoptogenic proteins such as cytochromec and AIF released from mitochondria upon permeability transition have been shown to directly induce cleavage of caspases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar,3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar). BetA-triggered mitochondrial PT and apoptosis involved cleavage of caspase-8 and caspase-3 independent of CD95 ligand/receptor interaction. Thus, in BetA-treated cells, caspase-8 became activated by mitochondria undergoing PT in the absence of CD95 death-inducing signaling complex formation. AIF released from mitochondria may mediate caspase-8 cleavage following BetA treatment since partially purified AIF could cleave recombinant caspase-8. In contrast, cytochromec did not induce activation of caspase-8, although it induced caspase-3 cleavage, indicating that the mechanism of caspase-8 activation downstream of mitochondria differed from that of caspase-3. Caspases might appear to be dispensable for mediating DNA fragmentation since partially purified AIF has been described to induce DNA fragmentation and since we found fragmentation of nuclei upon incubation with isolated mitochondria. However, these preparations may still contain small amounts of cytosolic fractions that may mediate activation of downstream targets such as PARP, DNA fragmentation factor, or caspase-activated deoxyribonuclease (44Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 45Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1997; 391: 43-50Crossref Scopus (2796) Google Scholar).Different classes of anticancer drugs may enter the apoptotic pathway at distinct entry sites before they eventually induce a Bcl-2/Bcl-XL-controlled ΔΨm disruption that marks the initiation of a common effector phase of apoptosis. Our findings may have implications for tumor therapy directed toward activation of apoptosis effector systems. Direct activation of mitochondrial PT, as exemplified by cytotoxic drugs such as BetA, may be sufficient for induction of apoptosis in cancer cells and may bypass the requirement for upstream signaling. Those drugs could still be effective against tumor cells that have a defect in upstream apoptosis pathways. In this context, it appears intriguing that tumors cells with a defect in the CD95 system (24Friesen C. Fulda S. Debatin K.-M. Leukemia (Baltimore). 1997; 1: 1833-1841Crossref Scopus (189) Google Scholar), which fail to respond to classical chemotherapeutic agents, are fully susceptible to BetA-induced cell death (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). Thus, our findings may be important to define and develop a new class of cytotoxic agents with direct mitochondrial effects that could overcome some forms of tumor-associated mechanisms of chemoresistance. Cytotoxic drugs have been reported to act primarily by inducing apoptosis in sensitive target cells (1Fisher D.E. Cell. 1994; 78: 539-542Abstract Full Text PDF PubMed Scopus (1368) Google Scholar). Triggering of apoptosis by anticancer drugs involves simultaneous or subsequent activation of death receptor systems, perturbation of mitochondrial function, and proteolytic processing of caspases, the death effector molecules of apoptosis (25Debatin K.-M. J. Natl. Cancer Inst. 1997; 89: 750-751Crossref PubMed Scopus (61) Google Scholar). Thus, the cell death pathway may be entered at multiple sites, and most drugs may hit various targets, although the precise molecular mechanisms have not been characterized in detail. Here, we report that one class of anticancer agents exemplified by BetA may act by directly targeting mitochondria, resulting in caspase activation downstream of mitochondria. Using a cell-free system, we found that BetA directly triggered PT in isolated mitochondria, and induction of PT appears to be the initial event in BetA-triggered apoptosis. Inhibition of PT by overexpression of Bcl-2 or Bcl-XL or by the mitochondrion-specific inhibitor BA prevented all manifestations of apoptosis in intact cells and in a cell-free system such as disruption of ΔΨm, activation of caspases, cleavage of substrates (PARP), and nuclear fragmentation. In contrast to BetA, classical cytotoxic drugs such as doxorubicin, cis-platinum, or etoposide did not induce mitochondrial perturbations in isolated mitochondria, suggesting that mitochondrial PT, which occurred in intact cells during apoptosis triggered by these substances (20Fulda S. Sieverts H. Friesen C. Herr I. Debatin K.-M. Cancer Res. 1997; 57: 3823-3829PubMed Google Scholar), was the consequence of a primary activation of other pathways or systems. BetA specifically kills neuroectodermal tumor cells (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). When added to intact cells, BetA specifically induced mitochondrial alterations in SHEP neuroblastoma cells, but not in lymphoid cell lines (data not shown). However, BetA-treated mitochondria isolated from the B lymphoblastoid cell line SKW6.4 triggered mitochondrial PT and mediated nuclear fragmentation, similar to mitochondria from SHEP cells. Thus, the specificity of BetA for neuroectodermal tumors may be explained by cell-specific uptake and/or translocation of the compound to the mitochondrial compartment rather than by differences in mitochondria themselves. In BetA-treated neuroblastoma cells, activation of different caspases and nuclear fragmentation were found only in cells with perturbed mitochondrial function, and BetA-induced loss of ΔΨm could not be inhibited by the caspase inhibitor Z-VAD-fmk. This suggests that caspase activation occurs downstream of mitochondria and that activation of mitochondria is sufficient to trigger all downstream events leading to apoptosis. In contrast, in doxorubicin-treated cells, loss of ΔΨm was inhibited by Z-VAD-fmk, indicating that activation of mitochondria is preceded by upstream caspase activation. 2Fulda, S., Susin, S. A., Kroemer, G., and Debatin, K.-M. (1998) Cancer Res. 58,4453–4460. Thus, BetA seems to define a class of cytotoxic agents whose apoptosis-inducing effect is predominately initiated by activation of mitochondria. Our studies on BetA-induced apoptosis provide a molecular sequence of the interplay between activation of different caspases and mitochondrial PT. Following death receptor triggering, the receptor proximal caspase-8 becomes activated, which in turn mediates full activation of the caspase cascade, cleavage of substrates, and concomitant triggering of mitochondrial PT (40Scaffidi C. Fulda S. Li F. Friesen C. Srinivasan A. Tomaselli K.J. Debatin K.-M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2617) Google Scholar, 41Medema J.P. Scaffidi C. Krammer P.H. Peter M.E. J. Biol. Chem. 1998; 273: 3388-3393Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 42Srinivasan A. Li F. Wong A. Kodandapani L. Smidt Jr., R. Krebs J.F. Fritz L.C. Wu J.C. Tomaselli K.J J. Biol. Chem. 1998; 273: 4523-4529Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 43Boise L.H. Thompson C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3759-3764Crossref PubMed Scopus (207) Google Scholar). Therefore, upon CD95 triggering, activation of caspase-8 occurs even in cells in which mitochondrial PT and processing of downstream caspases are blocked by overexpression of Bcl-2 or Bcl-XL (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar). In contrast, in BetA-treated cells, activation of both caspase-8 and caspase-3 was secondary to mitochondrial PT. Apoptogenic proteins such as cytochromec and AIF released from mitochondria upon permeability transition have been shown to directly induce cleavage of caspases (2Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1379) Google Scholar,3Susin S.A. Zamzami N. Castedo M. Daugas E. Wang H.G. Geley S. Fassy F. Reed J.C. Kroemer G. J. Exp. Med. 1997; 186: 5-37Crossref Scopus (587) Google Scholar, 6Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar). BetA-triggered mitochondrial PT and apoptosis involved cleavage of caspase-8 and caspase-3 independent of CD95 ligand/receptor interaction. Thus, in BetA-treated cells, caspase-8 became activated by mitochondria undergoing PT in the absence of CD95 death-inducing signaling complex formation. AIF released from mitochondria may mediate caspase-8 cleavage following BetA treatment since partially purified AIF could cleave recombinant caspase-8. In contrast, cytochromec did not induce activation of caspase-8, although it induced caspase-3 cleavage, indicating that the mechanism of caspase-8 activation downstream of mitochondria differed from that of caspase-3. Caspases might appear to be dispensable for mediating DNA fragmentation since partially purified AIF has been described to induce DNA fragmentation and since we found fragmentation of nuclei upon incubation with isolated mitochondria. However, these preparations may still contain small amounts of cytosolic fractions that may mediate activation of downstream targets such as PARP, DNA fragmentation factor, or caspase-activated deoxyribonuclease (44Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1638) Google Scholar, 45Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1997; 391: 43-50Crossref Scopus (2796) Google Scholar). Different classes of anticancer drugs may enter the apoptotic pathway at distinct entry sites before they eventually induce a Bcl-2/Bcl-XL-controlled ΔΨm disruption that marks the initiation of a common effector phase of apoptosis. Our findings may have implications for tumor therapy directed toward activation of apoptosis effector systems. Direct activation of mitochondrial PT, as exemplified by cytotoxic drugs such as BetA, may be sufficient for induction of apoptosis in cancer cells and may bypass the requirement for upstream signaling. Those drugs could still be effective against tumor cells that have a defect in upstream apoptosis pathways. In this context, it appears intriguing that tumors cells with a defect in the CD95 system (24Friesen C. Fulda S. Debatin K.-M. Leukemia (Baltimore). 1997; 1: 1833-1841Crossref Scopus (189) Google Scholar), which fail to respond to classical chemotherapeutic agents, are fully susceptible to BetA-induced cell death (37Fulda S. Friesen C. Los M. Scaffidi C. Mier W. Benedict M. Nuñez G. Krammer P.H. Peter M.E. Debatin K.-M. Cancer Res. 1997; 57: 4956-4964PubMed Google Scholar). Thus, our findings may be important to define and develop a new class of cytotoxic agents with direct mitochondrial effects that could overcome some forms of tumor-associated mechanisms of chemoresistance. We thank Didier Métivier (CNRS UPR420, Villejuif, France) for cytofluorometric analyses and Gabriel Nuñez and Mary Benedict (University of Michigan Medical School, Ann Arbor, MI) for SHEP-Neo, SHEP-Bcl-2, and SHEP-Bcl-XLtransfectants.

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.

Betulinic acid is a natural product with a range of biological effects, for example potent antitumor activity. This anticancer property is linked to its ability to induce apoptotic cell death in cancer cells by triggering the mitochondrial pathway of apoptosis. In contrast to the cytotoxicity of betulinic acid against a variety of cancer types, normal cells and tissue are relatively resistant to betulinic acid, pointing to a therapeutic window. Compounds that exert a direct action on mitochondria present promising experimental cancer therapeutics, since they may trigger cell death under circumstances in which standard chemotherapeutics fail. Thus, mitochondrion-targeted agents such as betulinic acid hold great promise as a novel therapeutic strategy in the treatment of human cancers.

Autophagy is a tightly-regulated catabolic process of cellular self-digestion by which cellular components are targeted to lysosomes for their degradation. Key functions of autophagy are to provide energy and metabolic precursors under conditions of starvation and to alleviate stress by removal of damaged proteins and organelles, which are deleterious for cell survival. Therefore, autophagy appears to serve as a pro-survival stress response in most settings. However, the role of autophagy in modulating cell death is highly dependent on the cellular context and its extent. There is an increasing evidence for cell death by autophagy, in particular in developmental cell death in lower organisms and in autophagic cancer cell death induced by novel cancer drugs. The death-promoting and -executing mechanisms involved in the different paradigms of autophagic cell death (ACD) are very diverse and complex, but a draft scenario of the key molecular targets involved in ACD is beginning to emerge. This review provides an up-to-date and comprehensive report on the molecular mechanisms of drug-induced autophagy-dependent cell death and highlights recent key findings in this exciting field of research.

The 'Individualized Therapy for Relapsed Malignancies in Childhood' (INFORM) precision medicine study is a nationwide German program for children with high-risk relapsed/refractory malignancies, which aims to identify therapeutic targets on an individualised basis. In a pilot phase, reported here, we developed the logistical and analytical pipelines necessary for rapid and comprehensive molecular profiling in a clinical setting. Fifty-seven patients from 20 centers were prospectively recruited. Malignancies investigated included sarcomas (n = 25), brain tumours (n = 23), and others (n = 9). Whole-exome, low-coverage whole-genome, and RNA sequencing were complemented with methylation and expression microarray analyses. Alterations were assessed for potential targetability according to a customised prioritisation algorithm and subsequently discussed in an interdisciplinary molecular tumour board. Next-generation sequencing data were generated for 52 patients, with the full analysis possible in 46 of 52. Turnaround time from sample receipt until first report averaged 28 d. Twenty-six patients (50%) harbored a potentially druggable alteration with a prioritisation score of 'intermediate' or higher (level 4 of 7). Common targets included receptor tyrosine kinases, phosphoinositide 3-kinase-mammalian target of rapamycin pathway, mitogen-activated protein kinase pathway, and cell cycle control. Ten patients received a targeted therapy based on these findings, with responses observed in some previously treatment-refractory tumours. Comparative primary relapse analysis revealed substantial tumour evolution as well as one case of unsuspected secondary malignancy, highlighting the importance of re-biopsy at relapse. This study demonstrates the feasibility of comprehensive, real-time molecular profiling for high-risk paediatric cancer patients. This extended proof-of-concept, with examples of treatment consequences, expands upon previous personalised oncology endeavors, and presents a model with considerable interest and practical relevance in the burgeoning era of personalised medicine.

Abstract Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.

Resveratrol, a naturally derived stilbene that exists in various foods and beverages, has attracted increasing attention over the past decade because of its multiple beneficial properties, including chemopreventive and antitumor activities. There are several other natural derivatives of resveratrol that are structurally similar to resveratrol and are also present in food. In addition, a series of resveratrol analogs have been synthesized by the addition of defined functional groups to increase the potency and/or enhance the activity of specific properties of resveratrol. Such resveratrol derivatives might provide promising tools as cancer chemopreventive agents, as well as cancer therapeutics in the prevention and treatment of cancer. This review provides an overview of key derivatives of resveratrol as cancer therapeutics.

Necroptosis represents a key programmed cell death pathway involved in various physiological and pathophysiological conditions. However, the role of reactive oxygen species (ROS) in necroptotic signaling has remained unclear. In the present study, we identify ROS as critical regulators of BV6/tumor necrosis factor-α (TNFα)-induced necroptotic signaling and cell death. We show that BV6/TNFα-induced cell death depends on ROS production, as several ROS scavengers such as butylated hydroxyanisole, N-acetylcysteine, α-tocopherol and ethyl pyruvate significantly rescue cell death. Before cell death, BV6/TNFα-stimulated ROS generation promotes stabilization of the receptor-interacting protein kinase 1 (RIP1)/RIP3 necrosome complex via a potential positive feedback loop, as on the one hand radical scavengers attenuate RIP1/RIP3 necrosome assembly and phosphorylation of mixed lineage kinase domain like (MLKL), but on the other hand silencing of RIP1 or RIP3 reduces ROS production. Although MLKL knockdown effectively decreases BV6/TNFα-induced cell death, it does not affect RIP1/RIP3 interaction and only partly reduces ROS generation. Moreover, the deubiquitinase cylindromatosis (CYLD) promotes BV6/TNFα-induced ROS generation and necrosome assembly even in the presence of BV6, as CYLD silencing attenuates these events. Genetic silencing of phosphoglycerate mutase 5 or dynamin-related protein 1 (Drp1) fails to protect against BV6/TNFα-induced cell death. By demonstrating that ROS are involved in regulating BV6/TNFα-induced necroptotic signaling, our study provides new insights into redox regulation of necroptosis.

Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death.

Programmed cell death via apoptosis is characteristically disturbed in human cancers. This facilitates not only tumor formation and progression, but also treatment resistance. Since many currently applied anticancer treatment strategies rely on intact cell death signaling pathways for their therapeutic efficacy, a better understanding of the regulatory mechanisms that control cell death signaling pathways is critical to bypass resistance. Thus, reactivation of cell death programs in cancer cells may open new perspectives for more effective and more tumor-selective, yet less toxic anticancer therapies.

Inhibitor of Apoptosis (IAP) proteins block programmed cell death and are expressed at high levels in various human cancers, thus making them attractive targets for cancer drug development. Second mitochondrial activator of caspases (Smac) mimetics are small-molecule inhibitors that mimic Smac, an endogenous antagonist of IAP proteins. Preclinical studies have shown that Smac mimetics can directly trigger cancer cell death or, even more importantly, sensitize tumor cells for various cytotoxic therapies, including conventional chemotherapy, radiotherapy, or novel agents. Currently, several Smac mimetics are under evaluation in early clinical trials as monotherapy or in rational combinations (i.e., GDC-0917/CUDC-427, LCL161, AT-406/Debio1143, HGS1029, and TL32711/birinapant). This review discusses the promise as well as some challenges at the translational interface of exploiting Smac mimetics as cancer therapeutics.

Ubiquitination of invading Salmonella Typhimurium triggers autophagy of cytosolic bacteria and restricts their spread in epithelial cells. Ubiquitin (Ub) chains recruit autophagy receptors such as p62/SQSTM1, NDP52/CALCOCO and optineurin (OPTN), which initiate the formation of double-membrane autophagosomal structures and lysosomal destruction in a process known as xenophagy. Besides this, the functional consequences and mechanistic regulation of differentially linked Ub chains at the host–Salmonella interface have remained unexplored. Here, we show, for the first time, that distinct Ub chains on cytosolic S. Typhimurium serve as a platform triggering further signalling cascades. By using single-molecule localization microscopy, we visualized the balance and nanoscale distribution pattern of linear (M1-linked) Ub chain formation at the surface of cytosolic S. Typhimurium. In addition, we identified the deubiquitinase OTULIN as central regulator of these M1-linked Ub chains on the bacterial coat. OTULIN depletion leads to enhanced formation of linear Ub chains, resulting in local recruitment of NEMO, activation of IKKα/IKKβ and ultimately NF-κB, which in turn promotes secretion of pro-inflammatory cytokines and restricts bacterial proliferation. Our results establish a role for the linear Ub coat around cytosolic S. Typhimurium as the local NF-κB signalling platform and provide insights into the function of OTULIN in NF-κB activation during bacterial pathogenesis. Deubiquitinase OTULIN targets linear (M1-linked) ubiquitin chain patches on cytosolic Salmonella Typhimurium to modulate NEMO, IKKα/IKKβ and NF-ĸB signalling and regulate secretion of pro-inflammatory cytokines and bacterial proliferation.

INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99-not applicable], compared with 117 days (95% CI, 106-143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases. SIGNIFICANCE: The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659.

Abstract Survivin is a member of the inhibitor of apoptosis proteins that is expressed at high levels in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Naturally occurring dietary compounds such as resveratrol have gained considerable attention as cancer chemopreventive agents. Here, we discovered a novel function of the chemopreventive agent resveratrol: resveratrol is a potent sensitizer of tumor cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through p53-independent induction of p21 and p21-mediated cell cycle arrest associated with survivin depletion. Concomitant analysis of cell cycle, survivin expression, and apoptosis revealed that resveratrol-induced G1 arrest was associated with down-regulation of survivin expression and sensitization for TRAIL-induced apoptosis. Accordingly, G1 arrest using the cell cycle inhibitor mimosine or induced by p21 overexpression reduced survivin expression and sensitized cells for TRAIL treatment. Likewise, resveratrol-mediated cell cycle arrest followed by survivin depletion and sensitization for TRAIL was impaired in p21- deficient cells. Also, down-regulation of survivin using survivin antisense oligonucleotides sensitized cells for TRAIL-induced apoptosis. Importantly, resveratrol sensitized various tumor cell lines, but not normal human fibroblasts, for apoptosis induced by death receptor ligation or anticancer drugs. Thus, this combined sensitizer (resveratrol)/inducer (e.g., TRAIL) strategy may be a novel approach to enhance the efficacy of TRAIL-based therapies in a variety of human cancers.

Abstract Whereas aberrant activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway, a key survival cascade, has previously been linked to poor prognosis in several human malignancies, its prognostic effect in neuroblastoma has not yet been explored. We therefore investigated the phosphorylation status of Akt, S6 ribosomal protein as target of mammalian target of rapamycin, and extracellular signal–regulated kinase (ERK) in 116 primary neuroblastoma samples by tissue microarray and its correlation with established prognostic markers and survival outcome. Here, we provide for the first time evidence that phosphorylation of Akt at serine 473 (S473) and/or threonine 308 (T308), S6 ribosomal protein, and ERK frequently occurs in primary neuroblastoma. Importantly, we identified Akt activation as a novel prognostic indicator of decreased event-free or overall survival in neuroblastoma, whereas phosphorylation of S6 ribosomal protein or ERK had no prognostic effect. In addition, Akt activation correlated with variables of aggressive disease, including MYCN amplification, 1p36 aberrations, advanced disease stage, age at diagnosis, and unfavorable histology. Monitoring Akt at T308 or both phosphorylation sites improved the prognostic significance of Akt activation in neuroblastoma specimens compared with S473 phosphorylation. Parallel experiments in neuroblastoma cell lines revealed that activation of Akt by insulin-like growth factor (IGF)-I significantly inhibited tumor necrosis factor–related apoptosis-inducing ligand– or chemotherapy-induced apoptosis in a PI3K-dependent manner because the PI3K inhibitor LY294002 completely reversed the IGF-I–mediated protection of neuroblastoma cells from apoptosis. By showing that activation of Akt correlates with poor prognosis in primary neuroblastoma in vivo and with apoptosis resistance in vitro, our findings indicate that Akt presents a clinically relevant target in neuroblastoma that warrants further investigation. [Cancer Res 2007;67(2):735–45]

Cytotoxic drugs commonly used in chemotherapy of leukemia and solid tumors have been shown to primarily act by inducing apoptosis in sensitive target cells. Apoptosis may involve activation of death-inducing ligand/receptor systems such as CD95 (APO-1/Fas). Treatment with anticancer drugs such as doxorubicin, methotrexate, cytarabine, etoposide and cisplatin at therapeutic concentrations leads to induction of CD95-ligand (CD95-L). CD95-L can mediate cell death in an autocrine/paracrine manner by crosslinking CD95 receptor (CD95). Interfering with CD95-ligand/receptor interaction by antagonistic antibodies to the receptor reduces sensitivity to drug-mediated apoptosis in some cell systems. In addition, treatment with cytotoxic drugs may result in upregulation of CD95, thereby increasing the sensitivity to the CD95 death signal. Apoptosis depends on activation of caspases. Deficient activation of the CD95 system was found in drug-resistant cells. In addition, CD95-resistant and doxorubicin-resistant cells displayed cross-resistance for induction of cell death. Thus, intact apoptosis pathways such as the CD95 system may play a role in determining sensitivity or resistance of tumor cells to chemotherapy.

Current attempts to improve the survival of cancer patients largely depend on strategies to target tumor cell resistance. Naturally occurring dietary compounds such as resveratrol have gained considerable attention as cancer chemopreventive agents. Here, we report that resveratrol acts as potent sensitizer for anticancer drug-induced apoptosis by inducing cell cycle arrest, which in turn resulted in survivin depletion. Concomitant analysis of cell cycle and apoptosis revealed that pretreatment with resveratrol resulted in cell cycle arrest in S phase and apoptosis induction preferentially out of S phase upon subsequent drug treatment. Likewise, cell cycle arrest in S phase by cell cycle inhibitors enhanced drug-induced apoptosis. Resveratrol-mediated cell cycle arrest sensitized for apoptosis by downregulating survivin expression through transcriptional and post-transcriptional mechanisms. Similarly, downregulation of survivin expression using survivin antisense oligonucleotides sensitized for drug-induced apoptosis. Importantly, downregulation of survivin and enhanced drug-induced apoptosis by resveratrol occurred in various human tumor cell lines irrespective of p53 status. Thus, this combined sensitizer (resveratrol)/inducer (cytotoxic drugs) concept may be a novel strategy to enhance the efficacy of anticancer therapy in a variety of human cancers.

Periplasmic proteins isolated by cold osmotic shock of Synechocystis sp. PCC 6803 cells were identified using 2D PAGE, MS and genome analysis. Most of the periplasmic proteins represent ‘hypothetical proteins’ with unknown function. A number of proteases of different specificity, and several enzymes involved in cell wall biosynthesis were also found. In salt‐adapted cells, six proteins were greatly enhanced and three proteins were newly induced. Most of the salt‐enhanced proteins are involved in the alteration of cell wall structure of salt‐adapted cells. The precursors of all 57 periplasmic proteins identified have a signal peptide; 47 of them contain a typical Sec‐dependent signal peptide, whereas 10 contain a putative twin‐arginine signal peptide.

Betulinic acid (BA) is a naturally occurring pentacyclic triterpene that exhibits a variety of biological activities including potent antitumor properties. This anticancer activity has been linked to its ability to directly trigger mitochondrial membrane permeabilization, a central event in the apoptotic process that seals the cell's fate. In contrast to the potent cytotoxicity of BA against a variety of cancer types, non-neoplastic cells as well as normal tissue remain relatively resistant to BA, thus pointing to a therapeutic window. Because agents that exert a direct action on mitochondria may bypass resistance to conventional chemotherapeutics, there is increasing interest to develop such compounds as experimental cancer therapeutics. Thus, mitochondrion-targeted agents such as BA hold great promise as a novel approach to overcome certain forms of drug resistance in human cancers.

Malignant brain tumors are the most common solid tumors in children. The overall prognosis for this group of patients is still poor, emphasizing the importance of more effective therapies. Betulinic acid (Bet A) has been described as a novel cytotoxic compound active against melanoma and neuroblastoma cells. Here we report that Bet A was active against medulloblastoma and glioblastoma cell lines. In addition, Bet A exerted cytotoxic activity against primary tumor cells cultured from patients in 4 of 4 medulloblastoma-tumor samples tested and in 20 of 24 glioblastoma-tumor samples. Since a small percentage of primary-glioblastoma-tumor cells (4/24) did not respond to Bet-A treatment, resistance to Bet A might occur. Induction of apoptosis by Bet A involved mitochondrial perturbations, since inhibition of the mitochondrial permeability transition by the mitochondrion-specific inhibitor bongkrekic acid (BA) reduced Bet-A-induced apoptosis. In addition, mitochondria undergoing Bet-A-induced permeability transition triggered DNA fragmentation in isolated nuclei. Cytochrome c was released from mitochondria of Bet-A-treated cells, and might be involved in activation of caspases. Following treatment with Bet A, caspase-8, caspase-3 and PARP were proteolytically processed. Inhibition of caspase cleavage by the broad-range caspase inhibitor zVAD.fmk strongly reduced Bet-A-induced apoptosis, indicating that apoptosis was mediated by activation of caspases. Since Bet A did not exhibit cytotoxicity against murine neuronal cells in vitro, these findings suggest that Bet A may be a promising new agent for the treatment of medulloblastoma and glioblastoma cells that clearly warrants further pre-clinical and clinical evaluation. Int. J. Cancer 82:435–441, 1999. © 1999 Wiley-Liss, Inc.

Suicide gene therapy systems such as the herpes simplex thymidine kinase/ganciclovir system (TK/GCV) may kill cancer cells by apoptosis through as yet undefined mechanisms. Here we show that TK/GCV treatment induces p53 accumulation and increases cell surface expression of CD95 and tumor necrosis factor receptor, which is likely to involve p53-mediated translocation of CD95 to the cell surface. TK/GCV-induced apoptosis involves CD95-L-independent CD95 aggregation leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing, death-inducing signaling complex. Dominant negative FADD, the caspase-8 inhibitor zIETD-fmk [Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone], and zVAD-fmk (Z-Val-Ala-Asp-fluoromethylketone) partially abrogate TK/GCV-induced apoptosis. In addition to apoptosis induction, TK/GCV treatment strongly sensitizes for CD95-L-, TNF-, and TNF-related, apoptosis-inducing, ligand (TRAIL)-induced cell death in constitutively resistant cells. These findings may be used to increase the efficacy of TK/GCV and other suicide gene therapy systems for the treatment of cancer.

Caloric restriction leads to retardation of the aging processes and to longer life in many organisms. This effect of caloric restriction can be mimicked by resveratrol, a natural plant product present in grapes and red wine, which is known as a potent activator of sirtuin 1 [silent mating type information regulation 2 homolog 1 (Sirt1)].One main effect of caloric restriction in mammals is a reduction of body fat from white adipose tissue. We sought to identify the effects of resveratrol on fat cell biology and to elucidate whether Sirt1 is involved in resveratrol-mediated changes.Human Simpson-Golabi-Behmel syndrome preadipocytes and adipocytes were used to study proliferation, adipogenic differentiation, glucose uptake, de novo lipogenesis, and adipokine secretion. Sirt1-deficient human preadipocytes were generated by using a lentiviral small hairpin RNA system to study the role of Sirt1 in resveratrol-mediated changes.Resveratrol inhibited preadipocyte proliferation and adipogenic differentiation in a Sirt1-dependent manner. In human adipocytes, resveratrol stimulated basal and insulin-stimulated glucose uptake. De novo lipogenesis was inhibited in parallel with a down-regulation of lipogenic gene expression. Furthermore, resveratrol down-regulated the expression and secretion of interleukin-6 and interleukin-8. Sirt1 was only partially responsible for the regulation of resveratrol-mediated changes in adipokine secretion.Taken together, our data suggest that resveratrol influences adipose tissue mass and function in a way that may positively interfere with the development of obesity-related comorbidities. Thus, our findings open up the new perspective that resveratrol-induced intracellular pathways could be a target for prevention or treatment of obesity-associated endocrine and metabolic adverse effects.

Natural products can exhibit many beneficial effects on human health. As far as cancer is concerned, naturally occurring compounds have been reported to prevent tumorigenesis and also to suppress the growth of established tumors. As cancer cells have evolved multiple mechanisms to resist the induction of programmed cell death (apoptosis), the modulation of apoptosis signaling pathways by natural compounds has been demonstrated to constitute a key event in these antitumor activities. This review presents some examples of how apoptosis pathways are targeted by selected naturally occurring agents and how these events can be exploited for cancer therapy.

Caspase-8 belongs to the caspase family of proteases and plays a key role in the regulation of programmed cell death (apoptosis) during normal development as well as in adult life. Since signaling via the death receptor (extrinsic) pathway critically depends on caspase-8, the disturbance of caspase-8 expression or function may contribute to human diseases. For example, caspase-8 is inactivated in a variety of human cancers, which may promote tumor progression as well as resistance to current treatment approaches. Therefore, caspase-8 presents a promising target to restore defective apoptosis programs in cancers in order to overcome resistance.

In the present study, changes in protein synthesis patterns after salt shock visualized by 35S-methionine labeling and the changed protein composition in salt-acclimated cells of the cyanobacterium Synechocystis sp. strain PCC 6803 were analyzed by a combination of 2-DE for protein separation and PMF for protein identification. As a basis for the differential analysis, a proteome map with 500 identified protein spots comprising 337 different protein species was established. Fifty-five proteins were found, which are induced by salt shock or accumulated after long-term salt acclimation. Some of the proteins are salt stress-specific, such as enzymes involved in the synthesis of the compatible solute glucosylglycerol, while most of them are involved in general stress acclimation. Particularly, heat-shock proteins and proteins acting against lesions by reactive oxygen species were found. Moreover, changes in enzymes involved in basic carbohydrate metabolism were detected. The dynamic of the proteome of salt-stressed Synechocystis cells was compared to previous data concerning transcriptome analysis revealing that 89% of the proteins induced shortly after salt shock were also found to be induced at the RNA level. However, 42% of the stably up-regulated proteins in salt-acclimated cells were not detected previously using DNA microarrays. The comparison of transcriptomic and proteomic analyses shows the significance of post-transcriptional regulatory mechanisms in acclimation of Synechocystis to high salt concentrations.

Killing of tumor cells by anticancer therapies commonly used in the treatment of cancer, e.g. chemotherapy, γ-irradiation, immunotherapy or suicide gene therapy, is predominantly mediated by triggering apoptosis, the cells intrinsic death program. Accordingly, defects in apoptosis pathways can result in cancer resistance to current treatment approaches. Understanding the molecular mechanisms that regulate cell death programs including apoptosis, and how resistant forms of cancer evade apoptotic events, may provide novel opportunities for cancer drug development. Keywords: apoptosis, cancer, mitochondria, cancer therapy, resistance

The plasma membrane of a cyanobacterial cell is crucial as barrier against the outer medium. It is also an energy-transducing membrane as well as essential for biogenesis of cyanobacterial photosystems and the endo-membrane system. Previously we have identified 57 different proteins in the plasma membrane of control cells from Synechocystis sp. strain PCC6803. In the present work, proteomic screening of salt-stress proteins in the plasma membrane resulted in identification of 109 proteins corresponding to 66 different gene products. Differential and quantitative analyses of 2-DE profiles of plasma membranes isolated from both control and salt-acclimated cells revealed that twenty proteins were enhanced/induced and five reduced during salt stress. More than half of the enhanced/induced proteins were periplasmic binding proteins of ABC-transporters or hypothetical proteins. Proteins that exhibited the highest enhancement during salt stress include FutA1 (Slr1295) and Vipp1 (Sll0617), which have been suggested to be involved in protection of photosystem II under iron deficiency and in thylakoid membrane formation, respectively. Other salt-stress proteins were regulatory proteins such as PII protein, LrtA, and a protein that belongs to CheY subfamily. The physiological significance of the identified salt-stress proteins in the plasma membrane is discussed integrating our current knowledge on cyanobacterial stress physiology.

Hypoxia inducible factor-1 (HIF-1) is the major transcription factor and key regulator of adoptive responses to hypoxia. Although it usually promotes tumor cell survival under hypoxia, it has also been implied to trigger apoptosis. Although the impact of hypoxia has been extensively studied in many adult solid tumors, its role in most childhood tumors, for example, in rhabdomyosarcoma (RMS) or Ewing sarcoma (ES), has not yet been addressed. Here, we report that hypoxia protects A204 RMS and A673 ES cells against anticancer drug- or tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and that Hif-1alpha plays a key role in conferring apoptosis resistance under hypoxia. Although a functional HIF-1 pathway and proapoptotic proteins such as p53 and Bcl-2/E1B 19 kDa interacting protein 3 were activated under hypoxia in both A204 RMS and A673 ES cells, these cells remained refractory to apoptosis. Concomitant analysis of antiapoptotic proteins revealed that hypoxia induced expression of Bcl-2 and inhibitor of apoptosis proteins (IAP)-2 as well as proteins associated with anaerobic metabolism such as the glucose transporter protein GLUT-1 and the glycolytic enzyme Aldolase A. Specific downregulation of Hif-1alpha by RNA interference significantly enhanced apoptosis under hypoxia by preventing the hypoxia-mediated increase in GLUT-1 expression without altering expression levels of the antiapoptotic proteins Bcl-2 or cIAP-2. Moreover, glucose deprivation-induced apoptosis of A204 RMS and A673 ES cells was inhibited under hypoxic conditions in a Hif-1alpha-dependent manner. As GLUT-1 was induced via Hif-1alpha under hypoxia in A204 RMS and A673 ES, these findings suggest that the Hif-1alpha-mediated increase in glucose uptake plays an important role in conferring apoptosis resistance. Thus, hypoxia-inducible genes may represent novel targets for therapeutic intervention in some pediatric tumors, which warrants further investigation.

One of the hallmarks of human cancers is the intrinsic or acquired resistance to apoptosis. Evasion of apoptosis can be part of a cellular stress response to ensure the cell's survival upon exposure to stressful stimuli. Apoptosis resistance may contribute to carcinogenesis, tumor progression, and also treatment resistance, since most current anticancer therapies including chemotherapy as well as radio- and immunotherapies primarily act by activating cell death pathways including apoptosis in cancer cells. Hence, a better understanding of the molecular mechanisms regarding how cellular stress stimuli trigger antiapoptotic mechanisms and how this contributes to tumor resistance to apoptotic cell death is expected to provide the basis for a rational approach to overcome apoptosis resistance mechanisms in cancers.

Background: There is mounting evidence in the literature that resveratrol is a promising natural compound for prevention and treatment of a variety of human cancers. This overview summarizes recent studies of the major apoptosis and survival pathways regulated by resveratrol. Biological mechanisms: Apoptosis or programmed cell death is a key regulator of tissue homeostasis during normal development and also in adult organism under various conditions including adaptive responses to cellular stress. For example, tissue homeostasis is maintained by tight control of signaling events regulating cell death and survival. Thus, uncontrolled proliferation or failure to undergo cell death is involved in pathogenesis and progression of many human diseases, for example in tumorigenesis or in cardiovascular disorders. Moreover, current cancer therapies primarily act by triggering apoptosis programs in cancer cells. Therapeutic applications: Natural products such as resveratrol have gained considerable attention as cancer chemopreventive or cardioprotective agents and also because of their antitumor properties. Among its wide range of biological activities, resveratrol has been reported to interfere with many intracellular signaling pathways, which regulate cell survival or apoptosis. Discussion: Further insights into the signaling network and interaction points modulated by resveratrol may provide the basis for novel drug discovery programs to exploit resveratrol for the prevention and treatment of human diseases.

Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.

Abstract Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer. [Cancer Res 2009;69(6):2425–34]

The aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway has been reported to correlate with adverse clinical outcome in human glioblastoma in vivo. However, the question of how this survival network can be successfully targeted to restore the sensitivity of glioblastoma to apoptosis induction has not yet been answered. Here, we report that inhibition of PI3K by LY294002 broadly sensitizes wild-type and mutant PTEN glioblastoma cells to both death receptor- and chemotherapy-induced apoptosis, whereas mammalian target of rapamycin (mTOR) inhibition is not sufficient to restore apoptosis sensitivity. LY294002 significantly enhances apoptosis triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), agonistic anti-CD95 antibodies, or several anticancer drugs (i.e., doxorubicin, etoposide, and vincristine) in a highly synergistic manner. In addition, LY294002 cooperates with TRAIL or doxorubicin to suppress colony formation, thus also showing a strong effect on long-term survival. Similarly, genetic knockdown of PI3K subunits p110alpha and/or p110beta by RNA interference (RNAi) primes glioblastoma cells for TRAIL- or doxorubicin-mediated apoptosis. In contrast to PI3K inhibition, pharmacologic or genetic blockade of mTOR by RAD001 (everolimus), rapamycin, or RNAi fails to enhance TRAIL- or doxorubicin-induced apoptosis. Analysis of apoptosis pathways reveals that PI3K inhibition acts in concert with TRAIL or doxorubicin to trigger mitochondrial membrane permeabilization, caspase activation, and caspase-dependent apoptosis, which are abolished by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Most importantly, PI3K inhibition by LY294002 sensitizes primary cultured glioblastoma cells obtained from surgical specimens to TRAIL- or chemotherapy-induced cell death. By showing that PI3K inhibition broadly primes glioblastoma cells for apoptosis, our findings provide the rationale for using PI3K inhibitors in combination regimens to enhance TRAIL- or chemotherapy-induced apoptosis in glioblastoma.

Abstract Betulinic acid (BA) is a naturally occurring pentacyclic triterpene that exhibits a variety of biological activities including potent antitumor properties. This anticancer activity has been linked to its ability to directly trigger mitochondrial membrane permeabilization, a central event in the apoptotic process that seals the cell's fate. In contrast to the potent cytotoxicity of BA against a variety of cancer types, nonmalignant cells and normal tissue remained relatively resistant to BA, indicating a therapeutic window. Since agents that exert a direct action on mitochondria may trigger cell death under circumstances in which standard chemotherapeutics fail, there is increasing interest to develop such compounds as experimental cancer therapeutics. Thus, mitochondrion‐targeted agents such as BA hold great promise as a novel approach to bypass certain forms of drug resistance in human cancers.

Abstract Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. Here, we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells, whereas they do not affect viability of normal peripheral blood lymphocytes, suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential, and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note, XIAP inhibitors even overcome Bcl-2–mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo, they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL.

Ferroptosis has recently been identified as a mode of programmed cell death. However, little is yet known about the signaling mechanism. Here, we report that lipoxygenases (LOX) contribute to the regulation of RSL3-induced ferroptosis in acute lymphoblastic leukemia (ALL) cells. We show that the glutathione (GSH) peroxidase 4 (GPX4) inhibitor RSL3 triggers lipid peroxidation, production of reactive oxygen species (ROS) and cell death in ALL cells. All these events are impeded in the presence of Ferrostatin-1 (Fer-1), a small-molecule inhibitor of lipid peroxidation. Also, lipid peroxidation and ROS production precede the induction of cell death, underscoring their contribution to cell death upon exposure to RSL3. Importantly, LOX inhibitors, including the selective 12/15-LOX inhibitor Baicalein and the pan-LOX inhibitor nordihydroguaiaretic acid (NDGA), protect ALL cells from RSL3-stimulated lipid peroxidation, ROS generation and cell death, indicating that LOX contribute to ferroptosis. RSL3 triggers lipid peroxidation and cell death also in FAS-associated Death Domain (FADD)-deficient cells which are resistant to death receptor-induced apoptosis indicating that the induction of ferroptosis may bypass apoptosis resistance. By providing new insights into the molecular regulation of ferroptosis, our study contributes to the development of novel treatment strategies to reactivate programmed cell death in ALL.

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP–BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP–BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

Programmed cell death is a basic cellular process that is critical to maintain tissue homeostasis. Besides apoptosis, necroptosis has more recently been discovered as another form of regulated cell death. Necroptosis plays a pivotal role during normal development and has also been implicated in the pathogenesis of a variety of human diseases. The control of necroptosis by defined signal transduction pathways offers the opportunity to target this cellular process for therapeutic purposes. For example, in cancer necroptosis is often impaired during tumorigenesis and can be engaged by targeted pharmacological approaches. Further insights into the signaling networks involved in the regulation of necroptosis will likely have important implications for the exploitation of this form of programmed cell death for the diagnosis or treatment of many diseases.

Abstract We have previously described novel histone acetyltransferase (HAT) inhibitors that block neuroblastoma cell growth in vitro . Here we show that two selected pyridoisothiazolone HAT inhibitors, PU139 and PU141, induce cellular histone hypoacetylation and inhibit growth of several neoplastic cell lines originating from different tissues. Broader in vitro selectivity profiling shows that PU139 blocks the HATs Gcn5, p300/CBP-associated factor (PCAF), CREB (cAMP response element-binding) protein (CBP) and p300, whereas PU141 is selective toward CBP and p300. The pan-inhibitor PU139 triggers caspase-independent cell death in cell culture. Both inhibitors block growth of SK-N-SH neuroblastoma xenografts in mice and the PU139 was shown to synergize with doxorubicin in vivo . The latter also reduces histone lysine acetylation in vivo at concentrations that block neoplastic xenograft growth. This is one of the very few reports on hypoacetylating agents with in vivo anticancer activity.

An easy and manageable in vitro screening system for drought tolerance of sunflower seedlings based on MS media supplemented with polyethylene glycol 6000 was evaluated. Morphological and physiological parameters were compared between control (-0.05 MPa) and drought-stressed (-0.6 MPa) seedlings of Helianthus annuus L. cv. Peredovick. There was a significant growth deficit in drought-stressed plants compared to control plants in terms of hypocotyl length, and shoot and root fresh mass. Shoot growth was more restricted than root growth, resulting in an increased root/shoot ratio of drought-stressed plants. Accumulation of osmolytes such as inositol (65-fold), glucose (58-fold), proline (55-fold), fructose (11-fold) and sucrose (eightfold), in leaves of drought-stressed plants could be demonstrated by gas-liquid chromatography. Soluble protein patterns of leaves were analysed with two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry. A set of 46 protein spots allowed identification of 19 marker proteins. Quantitative changes in protein expression of drought-stressed versus control plants were detected. In leaves of drought-stressed sunflower seedlings six proteins were significantly up-regulated more than twofold: a putative caffeoyl-CoA 3-O-methyltransferase (4.5-fold), a fructokinase 3 (3.3-fold), a vegetative storage protein (2.5-fold), a glycine-rich RNA binding protein (2.2-fold), a CuZn-superoxide dismutase (2.1-fold) and an unknown low molecular weight protein (2.3-fold). These proteins represent general stress proteins induced under drought conditions or proteins contributing to basic carbon metabolism. The up-regulated proteins are interesting candidates for further physiological and molecular investigations regarding drought tolerance in sunflower.

In this study, we report a novel mechanism of action for a cytotoxic derivative of betulinic acid (BA). B10 is a semi-synthetic glycosylated derivative of BA selected for its enhanced cytotoxic activity. Interestingly, although B10 induces apoptosis, caspase-3 downregulation incompletely prevents B10-induced cell death, Bcl-2 overexpression fails to protect cells and DNA fragmentation rates do not reflect cell death rates in contrast to cytoplasmic membrane permeabilization. These results implicate that apoptotic and non-apoptotic cell death coexist upon B10 treatment. Unexpectedly, we found that B10 induces autophagy and also abrogates the autophagic flux. B10 destabilizes lysosomes as shown by Lysotracker Red staining and by cathepsin Z and B release from lysosomes into the cytoplasm. Consistently, the cathepsin inhibitor Ca074Me significantly decreases B10-induced cell death, further supporting the fact that the release of lysosomal enzymes contributes to B10-triggered cell death. Downregulation of ATG7, ATG5 or BECN1 by RNAi significantly decreases caspase-3 activation, lysosomal permeabilization and cell death. Thus, by concomitant induction of autophagy and inhibition of the autophagic flux, B10 turns autophagy into a cell death mechanism. These findings have important implications for the therapeutic exploitation of BA derivatives, particularly in apoptosis-resistant cancers.

Abstract Cancer cells can be specifically driven into apoptosis by activating Death-receptor-4 (DR4; TRAIL-R1) and/or Death-receptor-5 (DR5; TRAIL-R2). Albeit showing promising preclinical efficacy, first-generation protein therapeutics addressing this pathway, especially agonistic anti-DR4/DR5-monoclonal antibodies, have not been clinically successful to date. Due to their bivalent binding mode, effective apoptosis induction by agonistic TRAIL-R antibodies is achieved only upon additional events leading to antibody-multimer formation. The binding of these multimers to their target subsequently leads to effective receptor-clustering on cancer cells. The research results presented here report on a new class of TRAIL-receptor agonists overcoming this intrinsic limitation observed for antibodies in general. The main feature of these agonists is a TRAIL-mimic consisting of three TRAIL-protomer subsequences combined in one polypeptide chain, termed the single-chain TRAIL-receptor–binding domain (scTRAIL-RBD). In the active compounds, two scTRAIL-RBDs with three receptor binding sites each are brought molecularly in close proximity resulting in a fusion protein with a hexavalent binding mode. In the case of APG350—the prototype of this engineering concept—this is achieved by fusing the Fc-part of a human immunoglobulin G1 (IgG1)-mutein C-terminally to the scTRAIL–RBD polypeptide, thereby creating six receptor binding sites per drug molecule. In vitro, APG350 is a potent inducer of apoptosis on human tumor cell lines and primary tumor cells. In vivo, treatment of mice bearing Colo205-xenograft tumors with APG350 showed a dose-dependent antitumor efficacy. By dedicated muteins, we confirmed that the observed in vivo efficacy of the hexavalent scTRAIL–RBD fusion proteins is—in contrast to agonistic antibodies—independent of FcγR-based cross-linking events. Mol Cancer Ther; 12(12); 2735–47. ©2013 AACR.

Autophagy represents a catabolic program involved in the degradation of cellular components via lysosomes. It serves to mitigate cellular stress and to provide metabolic precursors especially upon starvation. Thereby, autophagy can support the survival of cancer cells. In addition, there is now convincing evidence showing that under certain conditions autophagy can also foster cell death. This dual function of autophagy is also relevant upon anticancer treatment, as many chemotherapeutic agents engage autophagy. A better understanding of the molecular mechanisms that are critical for mediating autophagic cell death in cancer cells will be instrumental to selectively interfere with this cellular program in order to increase the cancer cell's response to cytotoxic drugs. This review illustrates how anticancer drug-induced autophagy is involved in mediating cell death.

Apoptosis is a form of programmed cell death that plays a critical role in the regulation of various physiological and pathophysiological processes. Since apoptosis is typically disturbed in human cancers, therapeutic targeting of apoptosis represents a promising avenue for the development of novel therapeutic approaches. This strategy is particularly relevant, since many currently used anticancer therapies utilize apoptosis signaling pathways to exert their antitumor activities. A better understanding of these signaling networks and their deregulation in human cancers is anticipated to open new perspectives for the development of apoptosis-targeted therapies for the treatment of cancer.

In most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A1 and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells.ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A1; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco's Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type.

Covalent modification of proteins with ubiquitin is essential for the majority of biological processes in mammalian cells. Numerous proteins are conjugated with single or multiple ubiquitin molecules or chains in a dynamic fashion, often determining protein half-lives, localization or function. Experimental approaches to study ubiquitination have been dominated by genetic and biochemical analysis of enzyme structure-function relationships, reaction mechanisms and physiological relevance. Here, we provide an overview of recent developments in microscopy-based imaging of ubiquitination, available reagents and technologies. We discuss the progress in direct and indirect imaging of differentially linked ubiquitin chains in fixed and living cells using confocal fluorescence microscopy and super-resolution microscopy, illustrated by the role of ubiquitin in antibacterial autophagy and pro-inflammatory signalling. Finally, we speculate on future developments and forecast a transition from qualitative to quantitative super-resolution approaches to understand fundamental aspects of ubiquitination and the formation and distribution of functional E3 ligase protein complexes in their native environment.

Abstract Necroptosis has recently been identified as an alternative form of programmed cell death that is characterized by defined molecular mechanisms. Reactive oxygen species (ROS) are involved in the regulation of numerous signaling pathways, as they are highly reactive and can cause (ir)reversible posttranslational modifications. While the role of ROS in other modes of cell death has been extensively studied, its impact on necroptotic signaling and cell death is far less clear. The current minireview discusses the evidence for and against a role of ROS in necroptosis.

Selective degradation of the endoplasmic reticulum (ER; reticulophagy) is a type of autophagy involved in the removal of ER fragments. So far, amino acid starvation as well as ER stress have been described as inducers of reticulophagy, which in turn restores cellular energy levels and ER homeostasis. Here, we explored the autophagy-inducing mechanisms that underlie the autophagic cell death (ACD)-triggering compound loperamide (LOP) in glioblastoma cells. Interestingly, LOP triggers upregulation of the transcription factor ATF4, which is accompanied by the induction of additional ER stress markers. Notably, knockout of ATF4 significantly attenuated LOP-induced autophagy and ACD. Functionally, LOP also specifically induces the engulfment of large ER fragments within autophagosomes and lysosomes as determined by electron and fluorescence microscopy. LOP-induced reticulophagy and cell death are predominantly mediated through the reticulophagy receptor RETREG1/FAM134B and, to a lesser extent, TEX264, confirming that reticulophagy receptors can promote ACD. Strikingly, apart from triggering LOP-induced autophagy and ACD, ATF4 is also required for LOP-induced reticulophagy. These observations highlight a key role for ATF4, RETREG1 and TEX264 in response to LOP-induced ER stress, reticulophagy and ACD, and establish a novel mechanistic link between ER stress and reticulophagy, with possible implications for additional models of drug-induced ER stress.Abbreviations: ACD: autophagic cell death; ATF6: activating transcription factor 6; ATL3: atlastin 3; BafA1: bafilomycin A1; CCPG1: cell cycle progression gene 1; co-IP: co-immunoprecipitation; DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; GBM: glioblastoma multiforme; HSPA5/BiP: heat shock protein family (Hsp70) member 5; LOP: loperamide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; RETREG1/FAM134B: reticulophagy regulator 1; RTN3L: reticulon 3 long; SEC62: SEC62 homolog, protein translocation factor; TEX264: testis-expressed 264, reticulophagy receptor; UPR: unfolded protein response.

Ligand binding triggers the oligomerization of TNFR1 monomers and dimers into trimers and higher-order oligomers.

Sterile inflammation is a therapeutic target in many diseases where it represents an important initiator of disease progression. However, the detailed mechanisms underlying its evolution and biological relevance are not yet completely elucidated. Biglycan, a prototype extracellular matrix-derived damage-associated molecular pattern, mediates sterile inflammation in macrophages through Toll-like receptor (TLR) 2 and/or TLR4-dependent signaling pathways. Here we discovered that soluble biglycan is a novel high-affinity ligand for CD14, a well-known GPI-anchored co-receptor for TLRs. CD14 is required for all biglycan-mediated TLR2/4 dependent inflammatory signaling pathways in macrophages. By binding to CD14 and choosing different TLR signaling branches, biglycan induced TNF-α and CCL2 via TLR2/4, HSP70 through TLR2, and CCL5 via TLR4. Mechanistically, biglycan evoked phosphorylation and subsequent nuclear translocation of p38, p44/42, and NF-κB, and these effects were due to a specific, high-affinity interaction between biglycan protein core and CD14. Finally, we provide proof-of-principle for the requirement of CD14, by transiently overexpressing biglycan in a mouse model of renal ischemia/reperfusion injury performed in Cd14−/− mice. Lack of Cd14 prevented biglycan-mediated cytokine expression, recruitment of macrophages, M1 macrophage polarization as well as mitigated the tubular damage and serum creatinine levels, thereby improving renal function. Thus, CD14 inhibition could lead to the reduction in the activation of biglycan-TLR2/4 signaling pathways and could be a novel therapeutic approach in inflammatory kidney diseases.

Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in ATG5 and ATG7 knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM.Abbreviations: ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.

We have identified the CD95 system as a key mediator of chemotherapy-induced apoptosis in leukemia and neuroblastoma cells. Here, we report that sensitivity of various solid tumor cell lines for drug-induced cell death corresponds to activation of the CD95 system. Upon drug treatment, strong induction of CD95 ligand (CD95-L) and caspase activity were found in chemosensitive tumor cells (Hodgkin, Ewing's sarcoma, colon carcinoma and small cell lung carcinoma) but not in tumor cells which responded poorly to drug treatment (breast carcinoma and renal cell carcinoma). Blockade of CD95 using F(ab')2 anti-CD95 antibody fragments markedly reduced drug-induced apoptosis, suggesting that drug-triggered apoptosis depended on CD95-L/receptor interaction. Moreover, drug treatment induced CD95 expression, thereby increasing sensitivity for CD95-induced apoptosis. Drug-induced apoptosis critically depended on activation of caspases (ICE/Ced-3-like proteases) since the broad-spectrum inhibitor of caspases zVAD-fmk strongly reduced drug-mediated apoptosis. The prototype substrate of caspases, poly(ADP-ribose) polymerase, was cleaved upon drug treatment, suggesting that CD95-L triggered autocrine/paracrine death via activation of caspases. Our data suggest that chemosensitivity of solid tumor cells depends on intact apoptosis pathways involving activation of the CD95 system and processing of caspases. Our findings may have important implications for new treatment approaches to increase sensitivity and to overcome resistance of solid tumors.

Recent evidence demonstrates that the anticancer activity of betulinic acid (BetA) can be markedly increased by combination protocols, for example with chemotherapy, ionizing radiation or TRAIL. Since nuclear factor-kappaB (NF-kappaB), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-kappaB in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-kappaB in a variety of tumor cell lines. NF-kappaB DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluorescence microscopy. BetA-induced NF-kappaB activation involved increased IKK activity and phosphorylation of IkappaB-alpha at serine 32/36 followed by degradation of IkappaB-alpha. Reporter assays revealed that NF-kappaB activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-kappaB activation by different chemical inhibitors (proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-kappaB inhibition by transient or stable expression of IkappaB-alpha super-repressor inhibited BetA-induced apoptosis in SH-EP neuroblastoma cells, while transient expression of IkappaB-alpha super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our findings that activation of NF-kappaB by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-kappaB inhibitors in combination with BetA would have no therapeutic benefit or could even be contraproductive in certain tumors, which has important implications for the design of BetA-based combination protocols.

We identified BetA as a new cytotoxic agent active against neuroectodermal tumor cells including neuroblastoma, medulloblastoma, glioblastoma and Ewing sarcoma cells, representing the most common solid tumors of childhood.BetA induced apoptosis by a direct effect on mitochondria independent of accumulation of wild-type p53 protein and independent of death-inducing ligand/receptor systems such as CD95. Mitochondrial perturbations on treatment with BetA resulted in the release of soluble apoptogenic factors such as cytochrome c or AIF from mitochondria into the cytosol, where they induced activation of caspases. Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L) that blocked loss of the mitochondrial membrane potential and cytochrome c release from mitochondria also conferred resistance to BetA. Most importantly, BetA exhibited potent antitumor activity on neuroblastoma cells resistant to CD95- or doxorubicin-triggered apoptosis and on primary tumor cells from patients with neuroectodermal tumors.Thus, BetA may be a promising new agent in the treatment of neuroectodermal tumors including neuroblastoma in vivo.

Chemotherapeutic agents and gamma-irradiation used in the treatment of brain tumors, the most common solid tumors of childhood, have been shown to act primarily by inducing apoptosis. Here, we report that activation of the CD95 pathway was involved in drug- and gamma-irradiation-induced apoptosis of medulloblastoma and glioblastoma cells. Upon treatment CD95 ligand (CD95-L) was induced that stimulated the CD95 pathway by crosslinking CD95 via an autocrine/paracrine loop. Blocking CD95-L/receptor interaction using F(ab')2 anti-CD95 antibody fragments strongly reduced apoptosis. Apoptosis depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3 like proteases) as it was almost completely abrograted by the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Apoptosis was mediated by cleavage of the receptor proximal caspase FLICE/MACH (caspase-8) and the downstream caspase CPP32 (caspase-3, Apopain) resulting in cleavage of the prototype caspase substrate PARP. Moreover, CD95 was upregulated in wild-type p53 cells thereby increasing responsiveness towards CD95 triggering. Since activation of the CD95 system upon treatment was also found in primary medulloblastoma cells ex vivo, these findings may have implications to define chemosensitivity and to develop novel therapeutic strategies in the management of malignant brain tumors.

Betulinic acid (BA), a natural component isolated from Birch trees, effectively induces apoptosis in neuroectodermal and epithelial tumor cells and exerts little toxicity in animal trials. Here, we show that BA-induced marked apoptosis in 65% of primary pediatric acute leukemia cells and all leukemia cell lines tested. When compared for in vitro efficiency with conventionally used cytotoxic drugs, BA was more potent than nine out of 10 standard therapeutics and especially efficient in tumor relapse. No crossresistances were found between BA and any cytotoxic drug. Intracellular apoptosis signaling in leukemia tumor cells paralleled the pathway found in neuroectodermal cells involving caspases, but not death receptors. In isolated mitochondria, BA induced release of both cytochrome c and Smac. Taken together, BA potently induces apoptosis in leukemia cells and should be further evaluated as a future drug to treat leukemia.

Because evasion of apoptosis can cause radioresistance of glioblastoma, there is a need to design rational strategies that counter apoptosis resistance. In the present study, we investigated the potential of targeting the antiapoptotic protein XIAP for the radiosensitization of glioblastoma. Here, we report that small-molecule XIAP inhibitors significantly enhance gamma-irradiation-induced loss of viability and apoptosis and cooperate with gamma-irradiation to suppress clonogenic survival of glioblastoma cells. Analysis of molecular mechanisms reveals that XIAP inhibitors act in concert with gamma-irradiation to cause mitochondrial outer membrane permeabilization, caspase activation, and caspase-dependent apoptosis. Importantly, XIAP inhibitors also sensitize primary cultured glioblastoma cells derived from surgical specimens as well as glioblastoma-initiating stemlike cancer stem cells for gamma-irradiation. In contrast, they do not increase the toxicity of gamma-irradiation on some nonmalignant cells of the central nervous system, including rat neurons or glial cells, pointing to some tumor selectivity. In conclusion, by demonstrating for the first time that small-molecule XIAP inhibitors increase the radiosensitivity of glioblastoma cells while sparing normal cells of the central nervous system, our findings build the rationale for further (pre)clinical development of XIAP inhibitors in combination with gamma-irradiation in glioblastoma.

Evasion of apoptosis can be caused by epigenetic silencing of caspase-8, a key component of the extrinsic apoptosis pathway. Loss of caspase-8 correlates with poor prognosis in medulloblastoma, which highlights the relevance of strategies to upregulate caspase-8 to break apoptosis resistance. Here, we develop a new combinatorial approach, that is treatment using histone deacetylase inhibitors (HDACI) together with interferon (IFN)-γ, to restore caspase-8 expression and to overcome resistance to the death-receptor ligand TNF-related apoptosis-inducing ligand (TRAIL) in medulloblastoma in vitro and in vivo. HDACI, for example, valproic acid (VA), suberoylanilide hydroxamic acid (SAHA) and MS-275, cooperate with IFN-γ to upregulate caspase-8 in cancer cells lacking caspase-8, thereby restoring sensitivity to TRAIL-induced apoptosis. Molecular studies show that VA promotes histone acetylation and acts in concert with IFN-γ to stimulate caspase-8 promoter activity. The resulting increase in caspase-8 mRNA and protein expression leads to enhanced TRAIL-induced activation of caspase-8 at the death-inducing signaling complex, mitochondrial outer-membrane permeabilization and caspase-dependent cell death. Intriguingly, pharmacological or genetic inhibition of caspase-8 also abolishes the VA/IFN-γ-mediated sensitization for TRAIL-induced apoptosis. It is important to note that VA and IFN-γ restore caspase-8 expression and sensitivity to TRAIL in primary medulloblastoma samples and significantly potentiate TRAIL-mediated suppression of medulloblastoma growth in vivo. These findings provide the rationale for further (pre)clinical evaluation of VA and IFN-γ to restore caspase-8 expression and apoptosis sensitivity in cancers with caspase-8 silencing and open new perspectives to overcome TRAIL resistance.

Abstract Disseminating tumors are one of the gravest medical problems. Here, we combine the tumor-specific apoptosis-inducing activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with the ability of mesenchymal stem cells (MSCs) to infiltrate both tumor and lymphatic tissues to target primary tumors as well as disseminated cancer cells in a human pancreatic cancer mouse model. Furthermore, we targeted X-linked inhibitor of apoptosis protein (XIAP) by RNA interference (RNAi) inside the cancer cells to make use of the apoptosis sensitization as well the antimetastatic effect that is afforded by XIAP silencing. We generated MSCs, termed MSC.sTRAIL, that express and secrete a trimeric form of soluble TRAIL (sTRAIL). MSC.sTRAIL triggered limited apoptosis in human pancreatic carcinoma cells that were resistant to soluble recombinant TRAIL, which is most likely due to the enhanced effect of the direct, cell-mediated delivery of trimeric TRAIL. MSC.sTRAIL-mediated cell death was markedly increased by concomitant knockdown of XIAP by RNAi in the cancer cells. These findings were confirmed in xenograft models, in which tumors from the parental pancreatic carcinoma cells showed only growth retardation on treatment with MSC.sTRAIL, whereas tumors with silenced XIAP that were treated with MSC.sTRAIL went into remission. Moreover, animals with XIAP-negative xenografts treated with MSC.sTRAIL were almost free of lung metastasis, whereas animals treated with control MSCs showed substantial metastatic growth in the lungs. In summary, this is the first demonstration that a combined approach using systemic MSC-mediated delivery of sTRAIL together with XIAP inhibition suppresses metastatic growth of pancreatic carcinoma.

Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.

A promising strategy in cancer therapy aims to promote apoptosis in cancer cells. Targeting inhibitor of apoptosis proteins (IAPs) with small-molecule inhibitors has attracted increasing interest in triggering cancer cell death. It is considered to have great potential for cancer drug discovery because IAPs block apoptosis at the core of the apoptotic machinery and are aberrantly expressed in various tumors. This review focuses on the current development of small-molecule IAP antagonists for cancer therapy.

Evasion from apoptotic cell death is reported to be a pivotal mechanism by which tumor cells acquire resistance to therapeutic treatment. Targeting the apoptotic pathways may constitute a promising strategy to counteract therapy resistance and to re-sensitize cancer cells. Expression of survivin, the smallest and structurally unique member of the inhibitor of apoptosis protein (IAP) family, has been shown to be associated with poor clinical outcome, more aggressive clinicopathologic features and resistance to both, conventional chemo and radiation therapy. Moreover, survivin detection in cancer tissue, in circulating tumor cells and in patient's serum has prognostic and predictive relevance and may display a prerequisite for marker based molecular therapies. Indeed, due to its universal over expression in malignant tissue, and its prominent role at disparate networks of cellular division, intracellular signaling, apoptosis and adaption to unfavorable surroundings, survivin has been shown to be a suitable target for a targeted therapy. The applicability of survivindriven strategies in clinical practice is currently under investigation as the first survivin antagonists (small molecule inhibitors, antisense oligonucleotides and immunotherapy) successfully entered phase I/II trials. Taken together, these data provide a rationale for the implementation of both, survivin as a molecular diagnostic tool and survivin targeted therapies, within future clinical practice.

The transcription factor CUX1 is known as a regulator of cell differentiation and cell cycle progression. Previously, CUX1 was identified as a modulator of invasiveness in various cancers. Based on expression profiles suggesting a role for CUX1 in mediating chemoresistance, the aim of this study was to characterise the effect of CUX1 on apoptosis as well as its regulation by signalling pathways modulating drug resistance in pancreatic cancer.The effect of CUX1 on TRAIL- (tumour necrosis factor-related apoptosis-inducing ligand) and drug-induced apoptosis was analysed using overexpression and knock-down strategies. Regulation of CUX1 by phosphatidylinositol-3-kinase (PI3K)/Akt signalling was examined at the mRNA and protein level. The effect of CUX1 knock-down by nanoparticle-complexed small interfering RNA (siRNA) in vivo was analysed in a murine xenograft model. Furthermore, CUX1 RNA and protein expression was evaluated in human pancreatic cancer and adjacent normal tissues.Knock-down of CUX1 resulted in significantly enhanced TRAIL- and drug-induced apoptosis, associated with increased PARP (poly ADP-ribose polymerase) cleavage and caspase activity. Vice versa, overexpression of CUX1 inhibited apoptosis. CUX1 expression was induced by activation of Akt/protein kinase B signalling, and decreased by PI3K inhibitors. The antiapoptotic effect of CUX1 was associated with upregulation of BCL2 and downregulation of tumour necrosis factor alpha. CUX1 was significantly overexpressed in pancreatic cancers, as analysed by in situ hybridisation and immunohistochemistry. In vivo, silencing of CUX1 by intratumourally administered polyethylenimine-complexed siRNA led to reduced tumour growth and increased apoptosis in pancreatic cancer xenografts.CUX1 was identified as an important mediator of tumour cell survival in pancreatic cancer in vitro and in vivo.

Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index <0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of cIAP1, leading to stabilization of NF-κB-inducing kinase and NF-κB activation. BV6-stimulated NF-κB activation is critically required for sensitization toward TMZ, as inhibition of NF-κB by overexpression of the mutant IκBα super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6-mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNFα) production. Also, TNFα, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6- and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-κB- and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ.

Background aims Cytokine-induced killer (CIK) cells may serve as an alternative approach to adoptive donor lymphocyte infusions (DLI) for patients with acute leukemia relapsing after haplo-identical hematopoietic stem cell transplantation (HSCT). We investigated the feasibility of enhancing CIK cell-mediated cytotoxicity by interleukin (IL)-15 against acute myeloid and lymphoblastic leukemia/lymphoma cells. Methods CIK cells were activated using IL-2 (CIKIL-2) or IL-15 (CIKIL-15) and phenotypically analyzed by fluorescence-activated cell sorting (FACS). Cytotoxic potential was measured by europium release assay. Results CIKIL-2 cells showed potent cytotoxicity against the T-lymphoma cell line H9, T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT-4 and subtype M4 acute myeloid leukemia (AML) cell line THP-1, but low cytotoxicity against the precursor B (pB)-cell ALL cell line Tanoue. IL-15 stimulation resulted in a significant enhancement of CIK cell-mediated cytotoxicity against acute lymphoblastic leukemia/lymphoma cell lines as well as against primary acute myeloid and defined lymphoblastic leukemia cells. However, the alloreactive potential of CIKIL-15 cells remained low. Further analysis of CIKIL-15 cells demonstrated that the NKG2D receptor is apparently involved in the recognition of target cells whereas killer-cell immunoglobulin-like receptor (KIR)-HLA mismatches contributed to a lesser extent to the CIKIL-15 cell-mediated cytotoxicity. In this context, CD3 + CD8 + CD25 + CD56− CIKIL-15 cell subpopulations were more effective in the lysis of AML cells, in contrast with CD56 + CIKIL-15 cells, which showed the highest cytotoxic potential against ALL cells. Conclusions This study provides the first evidence that CIKIL-15 cells may offer a therapeutic option for patients with refractory or relapsed leukemia following haplo-identical HSCT.

Evasion of programmed cell death represents one of the hallmarks of cancers that contributes to tumor initiation, progression and treatment resistance. This calls for novel therapeutic concepts to reactivate cell death programs in human malignancies. Since necroptosis represents a regulated form of necrosis that is under the control of defined signal transduction pathways, it offers molecular targets for rational therapeutic intervention. Indeed, there is mounting evidence showing that many currently used anticancer agents can engage necroptotic signaling pathways and thereby elicit cell death in malignant cells. A better understanding of the signaling networks regulating necroptosis in cancer cells is expected to speed up the development of anticancer drugs for therapeutic exploitation of necroptosis for cancer therapy.

Epigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-azacytidine (5-aza-CR, Vidaza) and 5-aza-2'-deoxycytidine (5-aza-dC, Dacogen) are the only clinically approved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These findings together with other reported differences enable and facilitate a rational design of new combination strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical oncology.

Inhibitor of apoptosis (IAP) proteins play a critical role in the control of survival and cell death by regulating key signaling events such as caspase activation and NF-κB signaling. Because aberrantly high expression of IAP proteins represents a frequent oncogenic event in human cancers, therapeutic targeting of IAP proteins is considered as a promising approach. Several small-molecule pharmacologic inhibitors of IAP proteins that mimic the binding domain of the endogenous IAP antagonist second mitochondrial activator of caspases (Smac) to IAP proteins have been developed over the past few years. IAP antagonists have been shown in various preclinical cancer models to either directly initiate cell death or, alternatively, to prime cancer cells for cytotoxic therapies by lowering the threshold for cell death induction. IAP antagonists (i.e., GDC-0917/CUDC-427, LCL161, AT-406, HGS1029, and TL32711) are currently under evaluation in early clinical trials alone or in combination regimens. Thus, the concept to therapeutically target IAP proteins in human cancer has in principle been successfully transferred into a clinical setting and warrants further evaluation as a treatment approach.

Searching for novel approaches to sensitize glioblastoma for cell death, we investigated the proteasome inhibitor bortezomib.The effect of bortezomib on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures, and in an in vivo model.Bortezomib and TRAIL synergistically trigger cell death and reduce colony formation of glioblastoma cells (combination index < 0.1). Investigations into the underlying molecular mechanisms reveal that bortezomib and TRAIL act in concert to cause accumulation of tBid, the active cleavage product of Bid. Also, the stability of TRAIL-derived tBid markedly increases on proteasome inhibition. Notably, knockdown of Bid significantly decreases bortezomib- and TRAIL-mediated cell death. By comparison, silencing of Noxa, which is also upregulated by bortezomib, does not confer protection. Coinciding with tBid accumulation, the activation of Bax/Bak and loss of mitochondrial membrane potential are strongly increased in cotreated cells. Overexpression of Bcl-2 significantly reduces mitochondrial perturbations and cell death, underscoring the functional relevance of the mitochondrial pathway. In addition, bortezomib cooperates with TRAIL to reduce colony formation of glioblastoma cells, showing an effect on long-term survival. Of note, bortezomib profoundly enhances TRAIL-triggered cell death in primary cultured glioblastoma cells and in patient-derived glioblastoma stem cells, underlining the clinical relevance. Importantly, bortezomib cooperates with TRAIL to suppress tumor growth in an in vivo glioblastoma model.These findings provide compelling evidence that the combination of bortezomib and TRAIL presents a promising novel strategy to trigger cell death in glioblastoma, including glioblastoma stem cells, which warrants further investigation.

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-κB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-κB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a physiological compound widely used in PDT as well as in tumor photodetection (PDD). Our results show that inhibition of NF-κB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-κB inhibition. We found that apoptosis was induced by PDT but in an incomplete manner and that, unexpectedly, NF-κB inhibition reduced its level. Oppositely PDT mainly induces necrosis in glioblastoma cells and NF-κB is found to have anti-necrotic functions in this context. The autophagic flux was also enhanced as a result of 5-ALA-PDT and we demonstrate that stimulation of autophagy acts as a pro-survival mechanism confering protection against PDT-mediated necrosis. These data point out that 5-ALA-PDT has an interesting potential as a mean to treat glioblastoma and that inhibition of NF-κB renders glioblastoma cells more sensitive to the treatment.

Glioblastoma is the most common primary brain tumor with a very poor prognosis, calling for novel treatment strategies. Here, we provide first evidence that histone deacetylase inhibitors (HDACI) prime glioblastoma cells for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis at least in part by c-myc-mediated downregulation of cellular FLICE-inhibitory protein (cFLIP). Pretreatment with distinct HDACI (MS275, suberoylanilide hydroxamic acid, valproic acid) significantly enhances TRAIL-induced apoptosis in several glioblastoma cell lines. Monitoring a panel of apoptosis-regulatory proteins revealed that MS275 reduces the expression of cFLIP(L) and cFLIP(S). This leads to decreased recruitment of cFLIP(L) and cFLIP(S) and increased activation of caspase-8 to the TRAIL death-inducing signaling complex, resulting in enhanced cleavage of caspase-8, -9 and -3 and caspase-dependent apoptosis. Also, MS275 promotes TRAIL-triggered processing of Bid, activation of Bax, loss of mitochondrial membrane potential and release of cytochrome c. MS275-mediated downregulation of cFLIP occurs at the mRNA level independent of proteasome- or caspase-mediated degradation, and is preceded by upregulation of nuclear levels of c-myc, a transcriptional repressor of cFLIP. Notably, MS275 causes increased binding of c-myc to the cFLIP promoter and reduces cFLIP promoter activity. Indeed, knockdown of c-myc partially rescues cFLIP(L) from MS275-inferred downregulation and significantly decreases TRAIL- and MS275-induced apoptosis. Also, overexpression of cFLIP(L) or cFLIP(S) significantly reduces MS275- and TRAIL-induced apoptosis. Importantly, MS275 sensitizes primary cultured glioblastoma cells towards TRAIL and cooperates with TRAIL to reduce long-term clonogenic survival of glioblastoma cells and to suppress glioblastoma growth in vivo underscoring the clinical relevance of this approach. Thus, these findings demonstrate that HDACI represent a promising strategy to prime glioblastoma for TRAIL-induced apoptosis by targeting cFLIP.

Recent genomic studies revealed a high rate of recurrent mutations in the RAS pathway in primary rhabdomyosarcoma (RMS) samples. In the present study, we therefore investigated how oncogenic RAS mutants impinge on the regulation of cell death of RMS13 cells. Here, we report that ectopic expression of NRAS12V, KRAS12V, or HRAS12V protects RMS13 cells from oxidative stress-induced cell death. RMS13 cells engineered to express NRAS12V, KRAS12V, or HRAS12V were significantly less susceptible to loss of cell viability upon treatment with several oxidative stress inducers including the thioredoxin reductase inhibitor Auranofin, the glutathione (GSH) peroxidase 4 inhibitor RSL3 or Erastin, an inhibitor of the cysteine/glutamate amino acid transporter system [Formula: see text] that blocks GSH synthesis. Notably, addition of Ferrostatin-1 confers protection against Erastin- or RSL3-induced cytotoxicity, indicating that these compounds trigger ferroptosis, an iron-dependent form of programed cell death. Furthermore, RMS13 cells overexpressing oncogenic RAS mutants are significantly protected against the dual PI3K/mTOR inhibitor PI103, whereas they are similarly sensitive to DNA-damaging drugs such as Doxorubicin or Etoposide. This suggests that oncogenic RAS selectively modulates cell death pathways triggered by cytotoxic stimuli in RMS13 cells. In conclusion, our discovery of an increased resistance to oxidative stress imposed by oncogenic RAS mutants in RMS13 cells has important implications for the development of targeted therapies for RMS.

On the basis of our previous identification of aberrant phosphatidylinositol-3-kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235-induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235-induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase-dependent apoptosis. Lysosome-mediated apoptosis occurs in a ROS-dependent manner, as ROS scavengers significantly reduce BEZ235/CQ-induced loss of MMP, LMP and apoptosis. There is a mitochondrial-lysosomal cross-talk, since lysosomal enzyme inhibitors significantly decrease BEZ235- and CQ-induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome-mediated apoptosis via a mitochondrial-lysosomal cross-talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor-based combination therapies.

The prognosis for patients with acute myeloid leukaemia (AML) is still poor, thus calling for novel treatment strategies. Here, we report that the small-molecule Smac mimetic BV6, which antagonizes Inhibitor of Apoptosis (IAP) proteins, acts in concert with cytarabine (AraC) to trigger cell death in AML cells in a highly synergistic manner (combination index 0.02–0.27). Similarly, BV6 cooperates with AraC to trigger cell death in primary AML samples, underscoring the clinical relevance of our findings. Molecular studies reveal that the TNFα-blocking antibody Enbrel significantly reduces BV6/AraC-induced cell death, demonstrating that an autocrine/paracrine TNFα loop mediates cell death. Furthermore, BV6 and AraC synergize to induce loss of mitochondrial membrane potential, caspase activation and DNA fragmentation, consistent with apoptotic cell death. Nevertheless, the caspase inhibitor zVAD.fmk fails to protect against BV6/AraC-induced cell death. Intriguingly, this cell death upon caspase inhibition is significantly reduced by pharmacological inhibition of two key components of necroptosis signaling, i.e. by RIP1 kinase inhibitor Necrostatin-1 or MLKL inhibitor NSA. Thus, BV6 sensitizes AML cells to AraC-induced cell death and overcomes apoptosis resistance by triggering necroptosis as alternative form of cell death. These findings have important implications for Smac mimetic-based strategies to bypass apoptosis resistance of AML.

We recently discovered mutation signatures reminiscent of BRCA deficiency in the vast majority of a set of primary osteosarcomas (OS).In the current study, we therefore investigated the sensitivity of a panel of OS cell lines to the poly(ADP)-ribose polymerase (PARP) inhibitor talazoparib alone and in combination with several chemotherapeutic drugs (i.e.temozolomide (TMZ), SN-38, doxorubicin, cisplatin, methotrexate (MTX), etoposide/carboplatin).Here, we identified an association between homologous recombination (HR) repair deficiency and the response of OS cell lines to talazoparib.All OS cell lines with molecular features characteristic of BRCA1/2 mutant tumors (so-called "BRCAness"), such as disruptive gains in PTEN or FANCD2 and/or losses of ATM, BAP1, BARD1 or CHEK2, were susceptible to talazoparib-induced reduction of cell viability (i.e.MG63, ZK-58,, SaOS-2 and MNNG-HOS).Consistent with their high sensitivity to talazoparib, MG63 and ZK-58 cells scored positive in a DNA-based measure of genomic instability (i.e.homologous recombination deficiency (HRD)-loss of heterozygosity (LOH) score).In contrast, U2OS cells that carry a heterozygous BRCA2 mutation and therefore most likely have one intact BRCA2 allele left proved to be resistant to talazoparib.Furthermore, we identified TMZ as the most potent chemotherapeutic drug together with talazoparib to synergistically reduce cell viability, as confirmed by calculation of combination index (CI) values, and to suppress long-term clonogenic survival.Mechanistically, talazoparib and TMZ cooperated to induce apoptotic cell death, as demonstrated by activation of BAX and BAK, loss of mitochondrial membrane potential (MMP), caspase activation, DNA fragmentation and caspase-dependent cell death.Genetic silencing of BAX and BAK or pharmacological inhibition of caspases by zVAD.fmksignificantly rescued OS cells from talazoparib/TMZ-induced apoptosis.These findings have important implications for the development of novel treatment strategies Research Paper Oncotarget 48795 www.impactjournals.com/oncotargetusing PARP inhibitors alone or together with chemotherapy in a subset of OS with features of BRCAness.

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.

Necroptosis contributes to the pathophysiology of several inflammatory, infectious and degenerative disorders. TNF-induced necroptosis involves activation of the receptor-interacting protein kinases 1 and 3 (RIPK1/3) in a necrosome complex, eventually leading to the phosphorylation and relocation of mixed lineage kinase domain like protein (MLKL). Using a high-content screening of small compounds and FDA-approved drug libraries, we identified the anti-cancer drug Sorafenib tosylate as a potent inhibitor of TNF-dependent necroptosis. Interestingly, Sorafenib has a dual activity spectrum depending on its concentration. In murine and human cell lines it induces cell death, while at lower concentrations it inhibits necroptosis, without affecting NF-κB activation. Pull down experiments with biotinylated Sorafenib show that it binds independently RIPK1, RIPK3 and MLKL. Moreover, it inhibits RIPK1 and RIPK3 kinase activity. In vivo Sorafenib protects against TNF-induced systemic inflammatory response syndrome (SIRS) and renal ischemia-reperfusion injury (IRI). Altogether, we show that Sorafenib can, next to the reported Braf/Mek/Erk and VEGFR pathways, also target the necroptotic pathway and that it can protect in an acute inflammatory RIPK1/3-mediated pathology.

Autophagy has long been thought to be an essential but unselective bulk degradation pathway. However, increasing evidence suggests selective autophagosomal turnover of a broad range of substrates. Bifunctional autophagy receptors play a key role in selective autophagy by tethering cargo to the site of autophagosomal engulfment. While the identity of molecular components involved in selective autophagy has been revealed at least to some extent, we are only beginning to understand how selectivity is achieved in this process. Here, we summarize the mechanistic and structural basis of receptor-mediated selective autophagy.

As inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the second mitochondrial activator of caspases (Smac) mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical nuclear factor-κB (NF-κB) signaling and a tumor necrosis factor-α (TNFα)/TNF receptor 1 (TNFR1) autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cellular IAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. Electrophoretic mobility shift assay supershift shows that the NF-κB DNA-binding subunits consist of p50, p52 and RelB further confirming the activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e., interleukin 8, monocyte chemoattractant protein 1, CXC chemokine receptor 4) and invasion (i.e., matrix metalloproteinase-9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have important implications for the use of Smac mimetics as cancer therapeutics.