From the Total Synthesis of Semi-Viriditoxin, Semi-Viriditoxic Acid and Dimeric Naphthopyranones to their Biological Activities in Burkitt B Cell Lymphoma.

Dimeric naphthopyranones are known to be biologically active, however, for the corresponding monomeric naphthopyranones this information is still elusive. Here the first enantioselective total synthesis of semi-viriditoxic acid as well as the synthesis of semi-viriditoxin and derivatives is reported. The key intermediate in the synthesis of naphthopyranones is an α,ß-unsaturated δ-lactone, which we synthesized in two different ways (Ghosez-cyclization and Grubbs ring-closing metathesis), while the domino-Michael-Dieckmann reaction of the α,ß-unsaturated δ-lactone with an orsellinic acid derivative is the key reaction. A structure-activity relationship study was performed measuring the cytotoxicity in Burkitt B lymphoma cells (Ramos). The dimeric structure was found to be crucial for biological activity: Only the dimeric naphthopyranones showed cytotoxic and apoptotic activity, whereas the monomers did not display any activity at all.

Cardiac injection of USSC boosts remuscularization of the infarcted heart by shaping the T-cell response.

Regenerating the injured heart remains one of the most vexing challenges in cardiovascular medicine. Cell therapy has shown potential for treatment of myocardial infarction, but low cell retention so far has limited its success. Here we show that intramyocardial injection of highly apoptosis-resistant unrestricted somatic stem cells (USSC) into infarcted rat hearts resulted in an unprecedented thickening of the left ventricular wall with cTnT+/BrdU+ cardiomyocytes that was paralleled by progressively restored ejection fraction. USSC induced significant T-cell enrichment in ischemic tissue with enhanced expression of T-cell related cytokines. Inhibition of T-cell activation by anti-CD28 monoclonal antibody, fully abolished the regenerative response which was restored by adoptive T-cell transfer. Secretome analysis of USSC and lineage tracing studies suggest that USSC secrete paracrine factors over an extended period of time which boosts a T-cell driven endogenous regenerative response mainly from adult cardiomyocytes.

An essential role of the autophagy activating kinase ULK1 in snRNP biogenesis.

The biogenesis of small uridine-rich nuclear ribonucleoproteins (UsnRNPs) depends on the methylation of Sm proteins catalyzed by the methylosome and the subsequent action of the SMN complex, which assembles the heptameric Sm protein ring onto small nuclear RNAs (snRNAs). In this sophisticated process, the methylosome subunit pICln (chloride conductance regulatory protein) is attributed to an exceptional key position as an 'assembly chaperone' by building up a stable precursor Sm protein ring structure. Here, we show that-apart from its autophagic role-the Ser/Thr kinase ULK1 (Uncoordinated [unc-51] Like Kinase 1) functions as a novel key regulator in UsnRNP biogenesis by phosphorylation of the C-terminus of pICln. As a consequence, phosphorylated pICln is no longer capable to hold up the precursor Sm ring structure. Consequently, inhibition of ULK1 results in a reduction of efficient UsnRNP core assembly. Thus ULK1, depending on its complex formation, exerts different functions in autophagy or snRNP biosynthesis.

The Impact of p70S6 Kinase-Dependent Phosphorylation of Gemin2 in UsnRNP Biogenesis.

The survival motor neuron (SMN) complex is a multi-megadalton complex involved in post-transcriptional gene expression in eukaryotes via promotion of the biogenesis of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). The functional center of the complex is formed from the SMN/Gemin2 subunit. By binding the pentameric ring made up of the Sm proteins SmD1/D2/E/F/G and allowing for their transfer to a uridine-rich short nuclear RNA (UsnRNA), the Gemin2 protein in particular is crucial for the selectivity of the Sm core assembly. It is well established that post-translational modifications control UsnRNP biogenesis. In our work presented here, we emphasize the crucial role of Gemin2, showing that the phospho-status of Gemin2 influences the capacity of the SMN complex to condense in Cajal bodies (CBs) in vivo. Additionally, we define Gemin2 as a novel and particular binding partner and phosphorylation substrate of the mTOR pathway kinase ribosomal protein S6 kinase beta-1 (p70S6K). Experiments using size exclusion chromatography further demonstrated that the Gemin2 protein functions as a connecting element between the 6S complex and the SMN complex. As a result, p70S6K knockdown lowered the number of CBs, which in turn inhibited in vivo UsnRNP synthesis. In summary, these findings reveal a unique regulatory mechanism of UsnRNP biogenesis.

NF90/NFAR (nuclear factors associated with dsRNA) - a new methylation substrate of the PRMT5-WD45-RioK1 complex.

Protein-arginine methylation is a common posttranslational modification, crucial to various cellular processes, such as protein-protein interactions or binding to nucleic acids. The central enzyme of symmetric protein arginine methylation in mammals is the protein arginine methyltransferase 5 (PRMT5). While the methylation reaction itself is well understood, recruitment and differentiation among substrates remain less clear. One mechanism to regulate the diversity of PRMT5 substrate recognition is the mutual binding to the adaptor proteins pICln or RioK1. Here, we describe the specific interaction of Nuclear Factor 90 (NF90) with the PRMT5-WD45-RioK1 complex. We show for the first time that NF90 is symmetrically dimethylated by PRMT5 within the RG-rich region in its C-terminus. Since upregulation of PRMT5 is a hallmark of many cancer cells, the characterization of its dimethylation and modulation by specific commercial inhibitors in vivo presented here may contribute to a better understanding of PRMT5 function and its role in cancer.

40 Years of Research on Polybrominated Diphenyl Ethers (PBDEs)-A Historical Overview and Newest Data of a Promising Anticancer Drug.

Polybrominated diphenyl ethers (PBDEs) are a group of molecules with an ambiguous background in literature. PBDEs were first isolated from marine sponges of Dysidea species in 1981 and have been under continuous research to the present day. This article summarizes the two research aspects, (i) the marine compound chemistry research dealing with naturally produced PBDEs and (ii) the environmental toxicology research dealing with synthetically-produced brominated flame-retardant PBDEs. The different bioactivity patterns are set in relation to the structural similarities and dissimilarities between both groups. In addition, this article gives a first structure-activity relationship analysis comparing both groups of PBDEs. Moreover, we provide novel data of a promising anticancer therapeutic PBDE (i.e., 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol; termed P01F08). It has been known since 1995 that P01F08 exhibits anticancer activity, but the detailed mechanism remains poorly understood. Only recently, Mayer and colleagues identified a therapeutic window for P01F08, specifically targeting primary malignant cells in a low µM range. To elucidate the mechanistic pathway of cell death induction, we verified and compared its cytotoxicity and apoptosis induction capacity in Ramos and Jurkat lymphoma cells. Moreover, using Jurkat cells overexpressing antiapoptotic Bcl-2, we were able to show that P01F08 induces apoptosis mainly through the intrinsic mitochondrial pathway.

Novel meriolin derivatives as rapid apoptosis inducers.

3-(Hetero)aryl substituted 7-azaindoles possessing multikinase inhibitor activity are readily accessed in a one-pot Masuda borylation-Suzuki coupling sequence. Several promising derivatives were identified as apoptosis inducers and, emphasizing the multikinase inhibition potential, as sphingosine kinase 2 inhibitors. Our measurements provide additional insights into the structure-activity relationship of meriolin derivatives, suggesting derivatives bearing a pyridine moiety with amino groups in 2-position as most active anticancer compounds and thus as highly promising candidates for future in vivo studies.

First Results from a Screening of 300 Naturally Occurring Compounds: 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol, 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol, and 5-epi-nakijinone Q as Substances with the Potential for Anticancer Therapy.

There is a variety of antineoplastic drugs that are based on natural compounds from ecological niches with high evolutionary pressure. We used two cell lines (Jurkat J16 and Ramos) in a screening to assess 300 different naturally occurring compounds with regard to their antineoplastic activity. The results of the compounds 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol (P01F03), 4,5,6-tribromo-2-(2',4'-dibromophenoxy)phenol (P01F08), and 5-epi-nakijinone Q (P03F03) prompted us to perform further research. Using viability and apoptosis assays on the cell lines of primary human leukemic and normal hematopoietic cells, we found that P01F08 induced apoptosis in the cell lines at IC50 values between 1.61 and 2.95 µM after 72 h. IC50 values of peripheral blood mononuclear cells (PBMNCs) from healthy donors were higher, demonstrating that the cytotoxicity in the cell lines reached 50%, while normal PBMNCs were hardly affected. The colony-forming unit assay showed that the hematopoietic progenitor cells were not significantly affected in their growth by P01F08 at a concentration of 3 µM. P01F08 showed a 3.2-fold lower IC50 value in primary leukemic cells [acute myeloid leukemia (AML)] compared to the PBMNC of healthy donors. We could confirm the antineoplastic effect of 5-epi-nakijinone Q (P03F03) on the cell lines via the induction of apoptosis but noted a similarly strong cytotoxic effect on normal PBMNCs.

Platelets induce apoptosis via membrane-bound FasL.

After tissue injury, both wound sealing and apoptosis contribute to restoration of tissue integrity and functionality. Although the role of platelets (PLTs) for wound closure and induction of regenerative processes is well established, the knowledge about their contribution to apoptosis is incomplete. Here, we show that PLTs present the death receptor Fas ligand (FasL) on their surface after activation. Activated PLTs as well as the isolated membrane fraction of activated PLTs but not of resting PLTs induced apoptosis in a dose-dependent manner in primary murine neuronal cells, human neuroblastoma cells, and mouse embryonic fibroblasts. Membrane protein from PLTs lacking membrane-bound FasL (FasL(△m/△m)) failed to induce apoptosis. Bax/Bak-mediated mitochondrial apoptosis signaling in target cells was not required for PLT-induced cell death, but increased the apoptotic response to PLT-induced Fas signaling. In vivo, PLT depletion significantly reduced apoptosis in a stroke model and an inflammation-independent model of N-methyl-d-aspartic acid-induced retinal apoptosis. Furthermore, experiments using PLT-specific PF4Cre(+) FasL(fl/fl) mice demonstrated a role of PLT-derived FasL for tissue apoptosis. Because apoptosis secondary to injury prevents inflammation, our findings describe a novel mechanism on how PLTs contribute to tissue homeostasis.

Cyclic Cystine-Bridged Peptides from the Marine Sponge Clathria basilana Induce Apoptosis in Tumor Cells and Depolarize the Bacterial Cytoplasmic Membrane.

Investigation of the sponge Clathria basilana collected in Indonesia afforded five new peptides, including microcionamides C (1) and D (2), gombamides B (4), C (5), and D (6), and an unusual amide, (E)-2-amino-3-methyl-N-styrylbutanamide (7), along with 11 known compounds, among them microcionamide A (3). The structures of the new compounds were elucidated by one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of the constituent amino acid residues in 1-7 were determined by Marfey's analysis. Microcionamides A, C, and D (1-3) showed in vitro cytotoxicity against lymphoma (Ramos) and leukemia cell lines (HL-60, Nomo-1, Jurkat J16), as well as against a human ovarian carcinoma cell line (A2780) with IC50 values ranging from 0.45 to 28 µM. Mechanistic studies showed that compounds 1-3 rapidly induce apoptotic cell death in Jurkat J16 and Ramos cells and that 1 and 2 potently block autophagy upon starvation conditions, thereby impairing pro-survival signaling of cancer cells. In addition, microcionamides C and A (1 and 3) inhibited bacterial growth of Staphylococcus aureus and Enterococcus faecium with minimal inhibitory concentrations between 6.2 and 12 µM. Mechanistic studies indicate dissipation of the bacterial membrane potential.

Autophagy signal transduction by ATG proteins: from hierarchies to networks.

Autophagy represents an intracellular degradation process which is involved in both cellular homeostasis and disease settings. In the last two decades, the molecular machinery governing this process has been characterized in detail. To date, several key factors regulating this intracellular degradation process have been identified. The so-called autophagy-related (ATG) genes and proteins are central to this process. However, several additional molecules contribute to the outcome of an autophagic response. Several review articles describing the molecular process of autophagy have been published in the recent past. In this review article we would like to add the most recent findings to this knowledge, and to give an overview of the network character of the autophagy signaling machinery.

Cleavage of annexin A1 by ADAM10 during secondary necrosis generates a monocytic "find-me" signal.

Annexin A1 is an intracellular calcium/phospholipid-binding protein that is involved in membrane organization and the regulation of the immune system. It has been attributed an anti-inflammatory role at various control levels, and recently we could show that annexin A1 externalization during secondary necrosis provides an important fail-safe mechanism counteracting inflammatory responses when the timely clearance of apoptotic cells has failed. As such, annexin A1 promotes the engulfment of dying cells and dampens the postphagocytic production of proinflammatory cytokines. In our current follow-up study, we report that exposure of annexin A1 during secondary necrosis coincided with proteolytic processing within its unique N-terminal domain by ADAM10. Most importantly, we demonstrate that the released peptide and culture supernatants of secondary necrotic, annexin A1-externalizing cells induced chemoattraction of monocytes, which was clearly reduced in annexin A1- or ADAM10-knockdown cells. Thus, altogether our findings indicate that annexin A1 externalization and its proteolytic processing into a chemotactic peptide represent final events during apoptosis, which after the transition to secondary necrosis contribute to the recruitment of monocytes and the prevention of inflammation.

Serum-derived plasminogen is activated by apoptotic cells and promotes their phagocytic clearance.

The elimination of apoptotic cells, called efferocytosis, is fundamentally important for tissue homeostasis and prevents the onset of inflammation and autoimmunity. Serum proteins are known to assist in this complex process. In the current study, we performed a multistep chromatographic fractionation of human serum and identified plasminogen, a protein involved in fibrinolysis, wound healing, and tissue remodeling, as a novel serum-derived factor promoting apoptotic cell removal. Even at levels significantly lower than its serum concentration, purified plasminogen strongly enhanced apoptotic prey cell internalization by macrophages. Plasminogen acted mainly on prey cells, whereas on macrophages no enhancement of the engulfment process was observed. We further demonstrate that the efferocytosis-promoting activity essentially required the proteolytic activation of plasminogen and was completely abrogated by the urokinase plasminogen activator inhibitor-1 and serine protease inhibitor aprotinin. Thus, our study assigns a new function to plasminogen and plasmin in apoptotic cell clearance.

Novel meriolin derivatives potently inhibit cell cycle progression and transcription in leukemia and lymphoma cells via inhibition of cyclin-dependent kinases (CDKs).

A key feature of cancer is the disruption of cell cycle regulation, which is characterized by the selective and abnormal activation of cyclin-dependent kinases (CDKs). Consequently, targeting CDKs via meriolins represents an attractive therapeutic approach for cancer therapy. Meriolins represent a semisynthetic compound class derived from meridianins and variolins with a known CDK inhibitory potential. Here, we analyzed the two novel derivatives meriolin 16 and meriolin 36 in comparison to other potent CDK inhibitors and could show that they displayed a high cytotoxic potential in different lymphoma and leukemia cell lines as well as in primary patient-derived lymphoma and leukemia cells. In a kinome screen, we showed that meriolin 16 and 36 prevalently inhibited most of the CDKs (such as CDK1, 2, 3, 5, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20). In drug-to-target modeling studies, we predicted a common binding mode of meriolin 16 and 36 to the ATP-pocket of CDK2 and an additional flipped binding for meriolin 36. We could show that cell cycle progression and proliferation were blocked by abolishing phosphorylation of retinoblastoma protein (a major target of CDK2) at Ser612 and Thr82. Moreover, meriolin 16 prevented the CDK9-mediated phosphorylation of RNA polymerase II at Ser2 which is crucial for transcription initiation. This renders both meriolin derivatives as valuable anticancer drugs as they target three different Achilles' heels of the tumor: (1) inhibition of cell cycle progression and proliferation, (2) prevention of transcription, and (3) induction of cell death.

Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2.

Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2-3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.

Pro-apoptotic and immunostimulatory tetrahydroxanthone dimers from the endophytic fungus Phomopsis longicolla.

Four tetrahydroxanthone dimers (1-4) and four biogenetically related monomers (5-8), including the new derivatives 4-6, were isolated from the endophyte Phomopsis longicolla. The absolute configurations of 2-4 were established for the first time by TDDFT electronic circular dichroism calculations, and that of phomoxanthone A (1) was revised by X-ray crystallography. Phomoxanthone A (1) showed the strongest pro-apoptotic activity when tested against a panel of human cancer cell lines, including cisplatin-resistant cells, whereas it was up to 100-fold less active against healthy blood cells. It was also the most potent activator of murine T lymphocytes, NK cells, and macrophages, suggesting an activation of the immune system in parallel to its pro-apoptotic activity. This dual effect in combating cancer cells could help in fighting resistance during chemotherapy. Preliminary structure-activity studies of isolated compounds and derivatives obtained by semisynthesis (9a-11) hinted at the location of the biaryl axis and the presence of acetyl groups as important structural elements for the biological activity of the studied tetrahydroxanthones.

Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

The spliceosome, responsible for all mature protein-coding transcripts of eukaryotic intron-containing genes, consists of small uridine-rich nuclear ribonucleoproteins (UsnRNPs). The assembly of UsnRNPs depends, on one hand, on the arginine methylation of Sm proteins catalyzed by the PRMT5 complex. On the other hand, it depends on the phosphorylation of the PRMT5 subunit pICln by the Uncoordinated Like Kinase 1 (ULK1). In consequence, phosphorylation of pICln affects the stability of the UsnRNP assembly intermediate, the so-called 6 S complex. The detailed mechanisms of phosphorylation-dependent integrity and subsequent UsnRNP assembly of the 6 S complex in vivo have not yet been analyzed. By using a phospho-specific antibody against ULK1-dependent phosphorylation sites of pICln, we visualize the intracellular distribution of phosphorylated pICln. Furthermore, we detect the colocaliphosphor-pICln1 with phospho-pICln by size-exclusion chromatography and immunofluorescence techniques. We also show that phosphorylated pICln is predominantly present in the 6 S complex. The addition of ULK1 to in vitro produced 6 S complex, as well as the reconstitution of ULK1 in ULK1-deficient cells, increases the efficiency of snRNP biogenesis. Accordingly, inhibition of ULK1 and the associated decreased pICln phosphorylation lead to accumulation of the 6 S complex and reduction in the spliceosomal activity of the cell.

Triggering of a novel intrinsic apoptosis pathway by the kinase inhibitor staurosporine: activation of caspase-9 in the absence of Apaf-1.

The protein kinase inhibitor staurosporine is one of the most potent and frequently used proapoptotic stimuli, although its mechanism of action is poorly understood. Here, we show that staurosporine as well as its analog 7-hydroxystaurosporine (UCN-01) not only trigger the classical mitochondrial apoptosis pathway but, moreover, activate an additional novel intrinsic apoptosis pathway. Unlike conventional anticancer drugs, staurosporine and UCN-01 induced apoptosis in a variety of tumor cells overexpressing the apoptosis inhibitors Bcl-2 and Bcl-x(L). Furthermore, activation of this novel intrinsic apoptosis pathway by staurosporine did not rely on Apaf-1 and apoptosome formation, an essential requirement for the mitochondrial pathway. Nevertheless, as demonstrated in caspase-9-deficient murine embryonic fibroblasts, human lymphoma cells, and chicken DT40 cells, staurosporine-induced apoptosis was essentially mediated by caspase-9. Our results therefore suggest that, in addition to the classical cytochrome c/Apaf-1-dependent pathway of caspase-9 activation, staurosporine can induce caspase-9 activation and apoptosis independently of the apoptosome. Since staurosporine derivatives have proven efficacy in clinical trials, activation of this novel pathway might represent a powerful target to induce apoptosis in multidrug-resistant tumor cells.

The incredible ULKs.

Macroautophagy (commonly abbreviated as autophagy) is an evolutionary conserved lysosome-directed vesicular trafficking pathway in eukaryotic cells that mediates the lysosomal degradation of intracellular components. The cytoplasmic cargo is initially enclosed by a specific double membrane vesicle, termed the autophagosome. By this means, autophagy either helps to remove damaged organelles, long-lived proteins and protein aggregates, or serves as a recycling mechanism for molecular building blocks. Autophagy was once invented by unicellular organisms to compensate the fluctuating external supply of nutrients. In higher eukaryotes, it is strongly enhanced under various stress conditions, such as nutrient and growth factor deprivation or DNA damage. The serine/threonine kinase Atg1 was the first identified autophagy-related gene (ATG) product in yeast. The corresponding nematode homolog UNC-51, however, has additional neuronal functions. Vertebrate genomes finally encode five closely related kinases, of which UNC-51-like kinase 1 (Ulk1) and Ulk2 are both involved in the regulation of autophagy and further neuron-specific vesicular trafficking processes. This review will mainly focus on the vertebrate Ulk1/2-Atg13-FIP200 protein complex, its function in autophagy initiation, its evolutionary descent from the yeast Atg1-Atg13-Atg17 complex, as well as the additional non-autophagic functions of its components. Since the rapid nutrient- and stress-dependent cellular responses are mainly mediated by serine/threonine phosphorylation, it will summarize our current knowledge about the relevant upstream signaling pathways and the altering phosphorylation status within this complex during autophagy induction.

Dual antitumour effect of 5-azacytidine by inducing a breakdown of resistance-mediating factors and epigenetic modulation.

BACKGROUND: The cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown promising anticancer activity in early clinical settings by selectively inducing apoptosis in different tumour types. However, some tumour entities such as hepatocellular carcinoma (HCC) display an inherent resistance to TRAIL. A huge effort has been made to unravel strategies for a clinically applicable sensitisation of resistant cancer cells to TRAIL. Reversible epigenetic alterations such as DNA methylation play a major role in development, maintenance and resistance phenomena of tumour cells. Currently, several clinical trials are exploiting the potential of epigenetic drugs, such as 5-azacytidine (5-aza-CR) or 5-aza-2'-deoxycytidine (5-aza-dC) to break primary or secondary resistance phenomena of cancer cells. Therefore, 5-aza-CR and 5-aza-dC were investigated in the context of TRAIL resistance. METHODS: Alterations in proliferation, apoptosis, regulatory proteins and toxicity were investigated in TRAIL-resistant hepatoma, and also in renal, colon and pancreatic cancer cells as well as non-transformed human-derived primary hepatocytes, tissue slices isolated from human liver and non-malignant colon cells, all of which had been exposed to demethylating drugs and/or TRAIL. RESULTS: Within hours, 5-aza-CR but not 5-aza-dC sensitised in vitro cultured tumour cells to TRAIL, first by activating caspases, followed by a subsequent induction of apoptosis. This surprisingly rapid sensitisation was confirmed in vivo employing a chorioallantoic membrane assay. As a major mechanism, a 5-aza-CR-induced inhibition of cellular protein synthesis was found which led to a breakdown of tumour-protecting factors such as the antiapoptotic factor FLICE inhibitory protein (FLIP). Importantly, TRAIL and 5-aza-CR did not induce relevant toxicity or apoptosis in primary hepatocytes, liver slices from different human donors and in normal colon cells. CONCLUSIONS: Molecular evidence is provided for a novel 5-aza-CR-based translational approach enabling a twofold treatment of apoptosis-resistant tumour entities, not only by an epigenetic reversion of the malignancy-associated phenotype but also by an efficient resensitization to apoptosis-inducing substances such as TRAIL.