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Increased FOXO3 nuclear localization is involved in neuroblastoma chemoresistance and tumor angiogenesis. Accordingly, FOXO3 inhibition is a promising strategy for boosting antitumor immune responses and suppressing FOXO3-mediated therapy resistance in cancer cells. However, no FOXO3 inhibitors are currently available for clinical use. Nevertheless, we have recently identified (4-propoxy)phenylpyrimidinylguanidine as a FOXO3 inhibitor in cancer cells in the low micromolar range. Here, we report the synthesis and structure-activity relationship study of a small library of its derivatives, some of which inhibit FOXO3-induced gene transcription in cancer cells in a submicromolar range and are thus 1 order of magnitude more potent than their parent compound. By NMR and molecular docking, we showed that these compounds differ in their interactions with the DNA-binding domain of FOXO3. These results may provide a foundation for further optimizing (4-propoxy)phenylpyrimidinylguanidine and developing therapeutics for inhibiting the activity of forkhead box (FOX) transcription factors and their interactions with other binding partners.

Conventional approaches in drug development involve testing on 2D-cultured mammalian cells, followed by experiments in rodents. Although this is the common strategy, it has significant drawbacks: in 2D cell culture with human cells, the cultivation at normoxic conditions on a plastic or glass surface is an artificial situation that significantly changes energy metabolism, shape and intracellular signaling, which in turn directly affects drug response. On the other hand, rodents as the most frequently used animal models have evolutionarily separated from primates about 100 million years ago, with significant differences in physiology, which frequently leads to results not reproducible in humans. As an alternative, spheroid technology and micro-organoids have evolved in the last decade to provide 3D context for cells similar to native tissue. However, organoids used for drug testing are usually just in the 50–100 micrometers range and thereby too small to mimic micro-environmental tissue conditions such as limited nutrient and oxygen availability. An attractive alternative offers 3D bioprinting as this allows fabrication of human tissue equivalents from scratch with hollow structures for perfusion and strict spatiotemporal control over the deposition of cells and extracellular matrix proteins. Thereby, tissue surrogates with defined geometry are fabricated that offer unique opportunities in exploring cellular cross-talk, mechanobiology and morphogenesis. These tissue-equivalents are also very attractive tools in drug testing, as bioprinting enables standardized production, parallelization, and application-tailored design of human tissue, of human disease models and patient-specific tissue avatars. This review, therefore, summarizes recent advances in 3D bioprinting technology and its application for drug screening.

Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In addition, basic and clinically oriented studies that investigate organ/tissue/cell dysfunction in various human diseases, including myopathies, cardiac/brain ischemia-reperfusion injuries, neurodegenerative diseases, cancer, and aging, require precise estimation of mitochondrial function. It should be noted that the main metabolic and functional characteristics of mitochondria obtained in situ (in permeabilized cells and tissue samples) and in vitro (in isolated organelles) are quite different, thereby compromising interpretations of experimental and clinical data. These differences are explained by the existence of the mitochondrial network, which possesses multiple interactions between the cytoplasm and other subcellular organelles. Metabolic and functional crosstalk between mitochondria and extra-mitochondrial cellular environments plays a crucial role in the regulation of mitochondrial metabolism and physiology. Therefore, it is important to analyze mitochondria in vivo or in situ without their isolation from the natural cellular environment. This review summarizes previous studies and discusses existing approaches and methods for the analysis of mitochondrial function, structure, and intracellular organization in situ.

FOXO transcription factors are critical regulators of cell homeostasis that control a plethora of different target genes and thereby steer cell death, differentiation, longevity and senescence in mammalian cells. The objective of the present study was to identify novel chemical compounds that bind to the DNA-binding domain of FOXO3 and modulate its transcriptional activity. The strategy was to combine pharmacophore-modelling-based in silico compound screening with fluorescence polarization protein-DNA binding assays and cell-based compound validation. By this approach, we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO transcription factors with highest affinity to human FOXO3. These compounds do not act as chemical inhibitors, but modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed by NMR spectroscopy and docking studies. We demonstrate that compound S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, modulate the transcription of distinct gene subsets and interfere with the physiological program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators by directly affecting protein-DNA interaction in normal and malignant cells.

Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired β-oxidation. Here, we performed lipidomic and proteomic analysis in fibroblasts from patients with very long-chain acyl-CoA dehydrogenase (VLCADD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies after incubation with heptanoate (C7) and octanoate (C8). Defects of β-oxidation induced striking proteomic alterations, whereas the effect of treatment with mc-FAs was minor. However, mc-FAs induced a remodeling of complex lipids. Especially C7 appeared to act protectively by restoring sphingolipid biosynthesis flux and improving the observed dysregulation of protein homeostasis in LCHADD under control conditions.

Objectives: Study of complex ROS dynamics and ROS mito-flashes in various living cells using fluorescent confocal imaging. Simultaneous analysis of the mitochondrial and extra-mitochondrial ROS dynamics. Analysis of the mitochondrial membrane potential and depolarization. Simultaneous analysis of mitochondrial calcium and ROS kinetics. It is well documented that mitochondria can produce a major amount of reactive oxygen species (ROS) which are involved in many physiological and pathological processes. The complex interrelationships, however, between ROS, mitochondrial inner membrane potential (\(\Delta\Psi\)m) and mitochondrial Ca2+ were not entirely investigated. In this work, we further underline biphasic ROS dynamics, demonstrating initial and continuing ROS expansion, followed by mitochondrial ROS flashes. Additionally, a huge heterogeneity in the rates of mitochondrial ROS production and ROS flashes start times has been shown. Comparing mitochondrial and extra mitochondrial fluorescence signals, we demonstrated that the mechanisms of ROS flashes may be a triggering of flashes by certain amounts of external ROS. These mechanisms involve mitochondrial permeability transition pore opening (collapse of mitochondrial membrane potential, \(\Delta\Psi\)m) and mitochondrial calcium sparks. Furthermore, mitochondria to mitochondria interactions can be seen as a wave propagation of mitochondrial ROS flashes and \(\Delta\Psi\)m collapses similar to the phenomenon of ROS-induced ROS release first demonstrated for cardiomyocytes. Our data show that mechanisms of mitochondrial ROS flashes activation and simultaneous depolarization can entail involvement of extra mitochondrial ROS produced either by individual mitochondrion or by adjacent mitochondria. This could represent common processes in ROS-ROS and mitochondria-mitochondria signaling, playing thus an important role in the cellular and mitochondrial physiology.

The transcription factor FOXO3 has been associated in different tumor entities with hallmarks of cancer, including metastasis, tumor angiogenesis, maintenance of tumor-initiating stem cells, and drug resistance. In neuroblastoma (NB), we recently demonstrated that nuclear FOXO3 promotes tumor angiogenesis in vivo and chemoresistance in vitro. Hence, inhibiting the transcriptional activity of FOXO3 is a promising therapeutic strategy. However, as no FOXO3 inhibitor is clinically available to date, we used a medium-throughput fluorescence polarization assay (FPA) screening in a drug-repositioning approach to identify compounds that bind to the FOXO3-DNA-binding-domain (DBD). Carbenoxolone (CBX), a glycyrrhetinic acid derivative, was identified as a potential FOXO3-inhibitory compound that binds to the FOXO3-DBD with a binding affinity of 19 µM. Specific interaction of CBX with the FOXO3-DBD was validated by fluorescence-based electrophoretic mobility shift assay (FAM-EMSA). CBX inhibits the transcriptional activity of FOXO3 target genes, as determined by chromatin immunoprecipitation (ChIP), DEPP-, and BIM promoter reporter assays, and real-time RT-PCR analyses. In high-stage NB cells with functional TP53, FOXO3 triggers the expression of SESN3, which increases chemoprotection and cell survival. Importantly, FOXO3 inhibition by CBX treatment at pharmacologically relevant concentrations efficiently repressed FOXO3-mediated SESN3 expression and clonogenic survival and sensitized high-stage NB cells to chemotherapy in a 2D and 3D culture model. Thus, CBX might be a promising novel candidate for the treatment of therapy-resistant high-stage NB and other “FOXO-resistant” cancers.

Identification of the mitochondrial roles and regulatory mechanisms for the understanding of muscle bioenergetics is a fundamental problem in physiology/pathophysiology. The three cytoskeletal systems microtubules, intermediate filaments, and microfilaments play a fundamental role in the maintenance of mitochondrial structure/architecture, intracellular organization and motility. Moreover, numerous communications between cytoskeleton and mitochondria can actively contribute to the regulation of mitochondrial respiratory function and oxidative phosphorylation (OXPHOS). In skeletal and cardiac muscles, mitochondrial positions are firmly fixed with very regular arrangement along filaments, providing a basis for many interactions with cellular systems such as cytoskeleton and sarcoplasmic reticulum (SR). Importantly, this might involve association of cytoskeletal elements with VDAC (the voltage-dependent anion channel), by this means, controlling the permeability of the outer mitochondrial membrane (OMM) and overall mitochondrial energy metabolism. Cardiac muscles and isolated cardiomyocytes show also a regular arrangement of tubulin beta-II fully co-localized with mitochondria, in contrast to other tubulin isoforms. This finding suggests the participation of tubulin beta-II in the regulation of OMM permeability through interaction with VDAC. In addition, the OMM permeability might be regulated by the specific isoform of cytoskeletal protein plectin. This book chapter summarizes and discusses previous studies on the role of cytoskeleton in the regulation of energy metabolism and mitochondrial physiology, OXPHOS and intracellular energy transfer.

Mitochondria play a key role in maintaining cell viability and cellular basic functions by energy (ATP) production. These organelles are also considered as an important source of reactive oxygen species (ROS) and several apoptosis activators. The crucial role of mitochondria has been shown in numerous aspects of cell physiology and pathophysiology, such as intracellular signaling. Mitochondria and mitochondrial function may deteriorate under pathological conditions like various diseases, ischemia-reperfusion (IR) injury and during aging. Damaged or injured mitochondria are the main causes for cell and tissue impairments, due to various cell stresses, generation of excess of ROS (oxidative stress), increased cellular and mitochondrial calcium levels, associated with apoptotic or/and necrotic cell death. The interplay among these mitochondrial activities under normal and pathological conditions is still unsuccessfully recognized. Mitochondria play a critical role in cardiac IR injury, where they are directly involved in various pathophysiological mechanisms. Here, we discuss the role of mitochondrial dynamics (fission, fusion) and heterogeneity. In particular, we stress the existence in the heart and skeletal muscles functionally different mitochondrial subpopulations that may also have different sensitivities to diseases and IR injury. Thus, different cardioprotective medications that maintain stability of mitochondria, their dynamics and turnover, including several agents, specific mitochondrial antioxidants, uncouplers, and application of ischemic preconditioning might be considered as the possible, beneficial strategies to protect mitochondria and cardiac function and improve longevity.

Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel—tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma—tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Background: D,L-Methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. Although the use of this opioid-receptor agonist as an anti-cancer drug is deemed controversial, this drug is gaining popularity in Europe as an “off-label” therapy despite insufficient research to support its benefits. In preclinical glioblastoma and leukaemia cell culture models D,L-methadone showed apoptotic and chemo-sensitizing effects. Its effects in ovarian cancer (OC) are completely unexplored. Methods: We analysed the OPRM mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 192 OC samples and 12 non-neoplastic control tissues. Pro-angiogenetic, cytotoxic and apoptotic effects of D,L-methadone were evaluated in OC cell-lines and four patient-derived tumor-spheroid models. Results: OPRM1 was transcriptionally expressed in 69% of OC-tissues and in five of eight OC cell-lines. D,L-methadone exposure significantly reduced cell-viability in five OC cell-lines irrespective of OPRM1 expression. D,L-methadone, applied alone or combined with cisplatin, showed no significant effects on apoptosis or VEGF secretion in cell-lines. Notably, in two of the four spheroid models, treatment with D,L-methadone resulted in significantly enhanced cell growth (by up to 121%), especially after long-term exposure. This is consistent with the observed attenuation of the inhibitory effects of cisplatin in three spheroid models when adding D,L-methadone. The effect of methadone treatment on VEGF secretion in tumor-spheroids was inconclusive. Conclusions: Our study demonstrates that certain OC samples express OPRM1, which, however, is not a prerequisite for D,L-methadone function. As such, D,L-methadone may exert detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect. Citation Format: Heidelinde Fiegl, Judith Hagenbuchner, Michael J. Ausserlechner, Christiana Kyvelidou, Beata Seeber, Sieghart Sopper, Irina Tsibulak, Verena Wieser, Julia Roessler, Elisabeth Reiser, Kaisa Huhtinen, Olli Carpén, Walther Parson, Christian Marth, Alain G. Zeimet. Effects of methadone on ovarian cancer cell-lines and patient-derived tumor-spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 947.

FOXO transcription factors are critical regulators of cell homeostasis and steer cell death, differentiation and longevity in mammalian cells. By combined pharmacophore-modeling-based in silico and fluorescence polarization-based screening we identified small molecules that physically interact with the DNA-binding domain (DBD) of FOXO3 and modulate the FOXO3 transcriptional program in human cells. The mode of interaction between compounds and the FOXO3-DBD was assessed via NMR spectroscopy and docking studies. We demonstrate that compounds S9 and its oxalate salt S9OX interfere with FOXO3 target promoter binding, gene transcription and modulate the physiologic program activated by FOXO3 in cancer cells. These small molecules prove the druggability of the FOXO-DBD and provide a structural basis for modulating these important homeostasis regulators in normal and malignant cells.

Inhibitor of apoptosis proteins (IAP) are cell death regulators that bind caspases and interfere with apoptotic signalling via death receptors or intrinsic cell death pathways. BIRC4/XIAP is the most potent anti-apoptotic IAP-member and it physically interacts with caspases via its BIR2 and its BIR3 domain. These domains are also critical for the interaction with mitochondria-derived SMAC/Diablo and with the IAP protein survivin. Survivin is frequently overexpressed in neuroblastoma due to a gain of 17q and we have demonstrated that survivin confers resistance to chemotherapeutic agents and reprograms metabolism of neuroblastoma cells towards glycolysis. As regulator of mitochondrial fission and autophagy survivin acts at the crossroads of mitochondrial architecture, autophagy and cellular energy metabolism. Methods: We tested the effect of SMAC-mimetics on the XIAP/survivin axis as modulator of cellular metabolism analysing mitochondrial morphology, metabolic intermediates and cellular survival. Finally, the impact of the combined treatment was evaluated in a xenograft neuroblastoma mouse model assessing the therapy effect on tumour size and volume. Results: Here we demonstrated that XIAP sequesters significant amounts of survivin within the cell that can be mobilized by so called SMAC-mimetics. SMAC-mimetics are drugs that are designed to bind with high affinity to XIAP-BIR2 / BIR3 domains to release caspases and re-sensitize XIAP-overexpressing tumors for chemotherapy. However, SMAC-mimetic treatment releases also survivin from XIAP and thereby induces mitochondrial fragmentation, prevents ROS accumulation and leads to the Warburg effect, an unwanted side effect of this therapy. Importantly, cells that drift into a highly glycolytic state due to SMAC-mimetic treatment become also highly sensitive to non-genotoxic treatment with glycolysis inhibitors such as 2-Deoxy-D-glucose (2DG) in vitro and in vivo. Conclusion: A combinational therapy of non-genotoxic SMAC-mimetics and glycolysis-inhibitors overcomes IAP-mediated cell survival in cancer and provides therefore an attractive usage of SMAC-mimetics.

Cancer cells bioenergetics is more dependent on glycolysis than mitochondrial oxidative phosphorylation, a phenomenon known as the Warburg Effect. It has been proposed that inhibition of glycolysis may selectively affect cancer cells. However, the effects of glycolysis inhibition on mitochondrial function and structure in cancer cells are not completely understood. Here, we investigated the comparative effects of 2-deoxy- d -glucose (2-DG, a glucose analogue, which suppresses cellular glycolysis) on cellular bioenergetics in human colon cancer DLD-1 cells, smooth muscle cells, human umbilical vein endothelial cells and HL-1 cardiomyocytes. In all cells, 2-DG treatment resulted in significant ATP depletion, however, the cell viability remained unchanged. Also, we did not observe the synergistic effects of 2-DG with anticancer drugs doxorubicin and 5-fluorouracil. Instead, after 2-DG treatment and ATP depletion, mitochondrial respiration and membrane potential were significantly enhanced and mitochondrial morphology changed in the direction of more network organization. Analysis of protein expression demonstrated that 2-DG treatment induced an activation of AMPK (elevated pAMPK/AMPK ratio), increased mitochondrial fusion (mitofusins 1 and 2) and decreased fission (Drp1) proteins. In conclusion, this study suggests a strong link between respiratory function and structural organization of mitochondria in the cell. We propose that the functionality of the mitochondrial network is enhanced compared to disconnected mitochondria.

// Judith Hagenbuchner 1, * , Martina Rupp 1, 2, * , Christina Salvador 2 , Bernhard Meister 2 , Ursula Kiechl-Kohlendorfer 1 , Thomas Muller 2 , Kathrin Geiger 3 , Consolato Sergi 4 , Petra Obexer 1, 3 , Michael J. Ausserlechner 2, 3 1 Departments of Pediatrics II, Medical University Innsbruck, Innsbruck, Austria 2 Pediatrics I, Medical University Innsbruck, Innsbruck, Austria 3 Tyrolean Cancer Research Institute, Innsbruck, Austria 4 Walter C. Mackenzie Centre, University of Alberta, Edmonton, Canada * These authors have contributed equally to this work Correspondence to: Michael J. Ausserlechner, email: Michael.J.Ausserlechner@i-med.ac.at Petra Obexer, email: Petra.Obexer@i-med.ac.at Keywords: chorioallantoic membrane (CAM), hypoxia, chemotherapy Received: June 07, 2016 Accepted: October 03, 2016 Published: October 18, 2016 ABSTRACT Neuroblastoma is the most frequent, extracranial solid tumor in children with still poor prognosis in stage IV disease. In this study, we analyzed FOXO3-phosphorylation and cellular localization in tumor biopsies and determined the function of this homeostasis regulator in vitro and in vivo . FOXO3-phosphorylation at threonine-32 (T32) and nuclear localization in biopsies significantly correlated with stage IV disease. DNA-damaging drugs induced nuclear accumulation of FOXO3, which was associated with elevated T32-phosphorylation in stage IV-derived neuroblastoma cells, thereby reflecting the in situ results. In contrast, hypoxic conditions repressed PKB-activity and caused dephosphorylation of FOXO3 in both, stroma-like SH-EP and high-stage-derived STA-NB15 cells. The activation of an ectopically-expressed FOXO3 in these cells reduced viability at normoxia, but promoted growth at hypoxic conditions and elevated VEGF-C-expression. In chorioallantoic membrane (CAM) assays STA-NB15 tumors with ectopic FOXO3 showed increased micro-vessel formation and, when xenografted into nude mice, a gene-dosage-dependent effect of FOXO3 in high-stage STA-NB15 cells became evident: low-level activation increased tumor-vascularization, whereas hyper-activation repressed tumor growth. The combined data suggest that, depending on the mode and intensity of activation, cellular FOXO3 acts as a homeostasis regulator promoting tumor growth at hypoxic conditions and tumor angiogenesis in high-stage neuroblastoma.

The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC50 value of 27.3 µg/mL (CI95: 25.9–28.9 µg/mL) corresponding to 9.6 µM (CI95: 9.1–10.1 µM) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR. The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5 ± 0.2 units (calculated average MW 2853.5) for the active fraction and 11.4 ± 0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6 ± 1.0 % active; 80.7 ± 2.7 % inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7″, 4–6″) and single linked B-type units. This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.

Transcription factors are central regulators of gene expression and critically steer development, differentiation and death. Except for ligand-activated nuclear receptors, direct modulation of transcription factor function by small molecules is still widely regarded as "impossible". This "un-druggability" of non-ligand transcription factors is due to the fact that the interacting surface between transcription factor and DNA is huge and subject to significant changes during DNA-binding. Besides some "success studies" with compounds that directly interfere with DNA binding, drug targeting approaches mostly address protein-protein interfaces with essential co-factors, transcription factor dimerization partners, chaperone proteins or proteins that regulate subcellular shuttling. An alternative strategy represent DNA-intercalating, alkylating or DNA-groove-binding compounds that either block transcription factor-binding or change the 3D-conformation of the consensus DNA-strand. Recently, much interest has been focused on chromatin reader proteins that steer the recruitment and activity of transcription factors to a gene transcription start site. Several small compounds demonstrate that these epigenetic reader proteins are exciting new drug targets for inhibiting lineage-specific transcription in cancer therapy. In this research update we will discuss recent advances in targeting transcription factors with small compounds, the challenges that are related to the complex function and regulation of these proteins and also the possible future directions and applications of transcription factor drug targeting.

Children diagnosed with Long-Chain-3-Hydroxy-Acyl-CoA-Dehydrogenase-Deficiency (LCHADD) or Very-Long-Chain-3-Hydroxy-Acyl-CoA-Dehydrogenase-Deficiency (VLCADD) frequently present with hypertrophic cardiomyopathy or muscle weakness which is caused by the accumulation of fatty acid metabolites due to inactivating mutations in the mitochondrial trifunctional protein. By analyzing mitochondrial morphology we uncovered that mutations within the HADHA or the ACADVL gene not only affect fatty acid oxidation, but also cause significant changes in the DNM1L/MFN2 ratio leading to the significant accumulation of truncated and punctate mitochondria in contrast to network-like mitochondrial morphology in controls. These striking morphological abnormalities correlate with changes in OXPHOS, an imbalance in ROS levels, reduced mitochondrial respiration, reduced growth rates and significantly increased glucose uptake per cell, suggesting that HADHA and ACADVL mutations shift cellular energy household into glycolysis. Experiments using the NOX2-specific inhibitor Phox-I2 suggest that NOX2 is activated by accumulating long-chain fatty acids and generates ROS, which in turn changes mitochondrial morphology and activity. We thereby provide novel insights into the cellular energy household of cells from LCHADD/VLCADD patients and demonstrate for the first time a connection between fatty acid metabolism, mitochondrial morphology and ROS in patients with these rare genetic disorders.

Adverse forms of neuroblastoma (NB), a childhood malignancy that develops from immature neuronal progenitor cells frequently carry a gain of chromosome 17q, which leads to overexpression of the antiapoptotic protein BIRC5/Survivin. We have recently shown that high Survivin expression shuts down mitochondrial complex I activity and shifts NB cells from oxidative phosphorylation to aerobic glycolysis, which further increases resistance to cell death induction. This increased glucose consumption sensitized tumor cells to glycolysis inhibitors. Interestingly, in Survivin-overexpressing cells 2-deoxy-d-glucose (2DG) treatment induces re-fusion of mitochondrial networks after 4 h, which coincides with Survivin repression. 2DG selectively acts on Survivin-expressing NB cells and induces autophagic degradation of Survivin via activation of the E3-ubiquitin ligase Parkin, a downstream target of PINK1. Survivin degradation further releases bound Beclin-1, which enhances autophagy and cell death induction. Knockdown of Parkin, however, reduces the sensitivity of Survivin-expressing NB cells to glycolysis inhibition. The selective activity of 2DG treatment on Survivin-overexpressing tumor cells was also confirmed in a xenograft mouse model, which further supports our hypothesis that glycolysis inhibitors might be useful drugs in the treatment of NB.

Forkhead box O class transcription factors are homeostasis regulators that control cell death, longevity and therapy-resistance. In neuroblastoma (NB), nuclear FOXO3 correlates with stage M disease and poor prognosis. To analyze whether FOXO3 contributes to drug-resistance in this childhood cancer, we investigated how different high-stage-derived NB cells respond to the activation of an ectopic FOXO3 allele. We found endogenous FOXO3 mostly localized to the nucleus-upon activation of an ectopic, 4OHT-activated FOXO3(A3)ER fusion protein two of the cell lines underwent apoptosis, whereas in the others FOXO3-activation even increased survival during drug-treatment. In the latter cell type, FOXO3 did not induce the BH3-only protein BCL2L11/BIM due to impaired binding of FOXO3 to the BIM-promoter, but still activated other FOXO3 targets. It was shown before that FOXO3 and TP53 physically interact with each other at two different regions-the TP53-N-terminus binds to the FOXO3-DNA binding domain (DBD) and the FOXO3-C-terminus interacts with the TP53-DBD. Interestingly, cell lines that undergo FOXO3-induced cell death carry homozygous point mutations in the TP53-DBD near the structural hotspot-mutation-site R175H, which abrogated FOXO3-TP53 interaction. In contrast, in FOXO3-death-resistant cells no point mutations in the TP53-DBD were found-in these cells FOXO3-TP53 complexes are formed and FOXO3-binding to the BIM-promoter, but not the induction of the detoxifying protein SESN3, were prevented, which in turn increased chemo-protection in this type of high-stage-derived NB cells. Our combined data suggest that FOXO3 steps in as a death inducer in case of TP53-mutation, whereas functional TP53 alters FOXO3-target-promoter-recognition, which prevents death induction by FOXO3 and instead increases chemo-protection and survival of NB cells. This novel mechanism may explain the low incidence of TP53 mutation in high-stage NB at diagnosis and suggests FOXO3 as a therapeutic target for this childhood malignancy.

// Judith Hagenbuchner 1 , Lorena Lungkofler 3 , Ursula Kiechl-Kohlendorfer 1 , Giampietro Viola 4 , Maria Grazia Ferlin 5 , Michael J. Ausserlechner 2 and Petra Obexer 1, 3 1 Department of Pediatrics II, Medical University Innsbruck, Innsbruck, Austria 2 Department of Pediatrics I, Medical University Innsbruck, Innsbruck, Austria 3 Tyrolean Cancer Research Institute, Innsbruck, Austria 4 Department of Woman’s and Child’s Health, Oncohematology Laboratory University of Padova, Padova, Italy 5 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy Correspondence to: Michael J. Ausserlechner, email: Michael.J.Ausserlechner@i-med.ac.at Petra Obexer, email: Petra.Obexer@i-med.ac.at Keywords: tubulin-inhibition, autophagy, NOXA, BCLXL, BIRC5/Survivin Received: June 21, 2016 Accepted: March 08, 2017 Published: March 22, 2017 ABSTRACT Neuroblastoma is the most frequent extra-cranial solid tumor in children with still high mortality in stage M. Here we studied the tubulin-inhibitor MG-2477 as a possible therapeutic agent for neuroblastoma therapy and uncovered that MG-2477 induces death in neuroblastoma cells independent of PKB-activation status and stage. MG-2477 triggers within 30 minutes extensive autophagosome-formation that finally leads to cell death associated with mitotic catastrophe. Autophagy is critical for MG-2477-induced death and is regulated by the BH3-only protein PMAIP1/NOXA which sequesters the anti-apoptotic BCL2-protein BCLXL and thereby displaces and activates the autophagy-regulator BECN1/beclin1. Knockdown of NOXA or overexpression of its pro-survival binding partners MCL1 and BCLXL counteracts MG-2477-induced cell death. MG-2477 also rapidly induces the repression of the anti-apoptotic protein Survivin, which promotes autophagy and cell death. We further observed the accumulation of reactive oxygen species (ROS) that triggers autophagy induction suggesting a change of the PI3 kinase-III/BECN1 complex and activates the transcription factor FOXO3, which contributes to final cell death induction. The combined data suggest that MG-2477 induces a sequential process of ROS-accumulation, autophagy and FOXO3-activation that leads to cell death in neuroblastoma cells.

The transcription factor FOXO3 is associated with poor outcome in high-stage neuroblastoma (NB), as it facilitates chemoprotection and tumor angiogenesis. In other tumor entities, FOXO3 stimulates metastasis formation, one of the biggest challenges in the treatment of aggressive NB. However, the impact of FOXO3 on the metastatic potential of neuronal tumor cells remains largely unknown. In the present study, we uncover the small leucine-rich proteoglycan family member lumican (LUM) as a FOXO3-regulated gene that stimulates cellular migration in NB. By a drug-library screen we identified the small molecular weight compound repaglinide (RPG) as a putative FOXO3 inhibitor. Here, we verify that RPG binds to the FOXO3-DNA-binding-domain (DBD) and thereby silences the transcriptional activity of FOXO3. Consistent with the concept that the FOXO3/LUM axis enhances the migratory capacity of aggressive NB cells, we demonstrate that stable knockdown of LUM abrogates the FOXO3-mediated increase in cellular migration. Importantly, FOXO3 inhibition by RPG represses the binding of FOXO3 to the LUM promoter, inhibits FOXO3-mediated LUM RNA and protein expression, and efficiently abrogates FOXO3-triggered cellular &ldquo, wound healing&rdquo, as well as spheroid-based 3D-migration. Thus, silencing the FOXO3/LUM axis by the FDA-approved compound RPG represents a promising strategy for novel therapeutic interventions in NB and other FOXO3-dependent tumors.

// Judith Hagenbuchner 1,3 , Ursula Kiechl-Kohlendorfer 1 , Petra Obexer 1,3 , Michael J. Ausserlechner 2,3 1 Department of Pediatrics II, Medical University Innsbruck, Austria 2 Department of Pediatrics I, Medical University Innsbruck, Austria 3 Tyrolean Cancer Research Institute, Innsbruck, Austria Correspondence: Petra Obexer, email: // Michael Ausserlechner, email: // Keywords : Mcl1L, apoptosis, BCL2 proteins, mRNA variant Received : July 1, 2013 Accepted : July 13, 2013 Published : July 15, 2013 Abstract Members of the Bcl-2 protein family are frequently deregulated in tumors as they critically control cell death induction in mammalian cells. Alterations of these proteins may cause resistance to chemotherapy-induced cell death and immune responses. By serendipity we cloned a variant of the anti-apoptotic Bcl2-family member Myeloid cell leukemia-1 (Mcl1) from human neuroblastoma and leukemia cells. This Mcl1L variant lacks a 45 bp sequence that codes for 15 highly conserved amino acids ranging from Gly158 to Asp172. This region is part of the so called PEST-sequence of Mcl1L and contains two phosphorylation sites (Ser159 and Thr163) that regulate Mcl1L stability. A caspase 3/caspase 8 cleavage site at Asp157 which has been reported to be critical for death-receptor-induced apoptosis and for the conversion of Mcl1L into a pro-apoptotic protein is also missing in this novel variant. Importantly, Mcl1L delGly158-Asp172 bound significantly more pro-apoptotic Bim compared to Mcl1L and showed increased anti-proliferative and anti-apoptotic activity compared to Mcl1L during death receptor-induced cell death. This suggests that this novel Mcl1L variant efficiently protects tumor cells against extrinsic death signalling and therefore may provide a survival advantage for highly aggressive tumors.

Background Neuroblastoma is the most common solid tumor in childhood and develops from undifferentiated progenitor cells of the sympathetic nervous system. In neuronal tumor cells DNA-damaging chemotherapeutic agents activate the transcription factor FOXO3 which regulates the formation of reactive oxygen species (ROS) and cell death as well as a longevity program associated with therapy resistance. We demonstrated before that C10ORF10/DEPP, a transcriptional target of FOXO3, localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification. In the present study, we investigated the impact of FOXO3 and DEPP on the regulation of autophagy. Autophagy serves to reduce oxidative damage as it triggers a self-degradative process for the removal of aggregated or misfolded proteins and damaged organelles. Methods The effect of FOXO3 and DEPP on autophagy induction was analyzed using live cell fluorescence microscopy and immunoblot analyses of SH-EP cells transfected with a plasmid for EYFP-LC3 and with siRNAs specific for LC3, respectively. ROS steady-state levels were measured with reduced MitoTrackerRed CM-H2XROS. Cellular apoptosis was analyzed by flow cytometry and the caspase 3/7 assay. Results We report for the first time that DEPP induces ROS accumulation and thereby mediates the formation of autophagosomes as inhibition of ROS formation by N-acetyl-cysteine completely blocks autophagy. We further demonstrate that H2O2-treatment triggers autophagy-induction by FOXO3-mediated DEPP expression. Importantly, knockdown of DEPP was sufficient to efficiently inhibit autophagy-induction under different stress conditions such as serum starvation and genotoxic stress, suggesting that DEPP expression is critical for the initiation of autophagy in neuroblastoma. FOXO3-triggered autophagy partially protects neuroblastoma cells from cell death. Consistent with this concept, we demonstrate that inhibition of autophagy by LC3-knockdown significantly increased etoposide- and doxorubicin-induced apoptosis. These results were also confirmed by the use of the autophagy-inhibitor chloroquine that significantly enhanced the chemotherapeutic effect of etoposide and doxorubicin in neuronal tumor cells. Conclusion Targeting FOXO3/DEPP-triggered autophagy is a promising strategy to sensitize neuroblastoma cells to chemotherapy. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0661-4) contains supplementary material, which is available to authorized users.

Gain of chromosome 17q correlates with high-stage disease, an adverse clinical outcome and leads to the overexpression of the antiapoptotic protein BIRC5/Survivin in neuroblastoma (NB). We have shown before that Survivin defines a threshold for the sensitivity of NB cells to DNA-damaging chemotherapeutic agents that require FOXO3 activation for apoptosis induction. To investigate the molecular basis of apoptosis inhibition we analyzed the function of Survivin at mitochondria and uncovered that Survivin induces mitochondrial fragmentation, reduces mitochondrial respiration and represses BCL2L11/Bim. Mitochondrial fission depends on Survivin-induced recruitment of the fission regulator DNM1L/Drp1 to mitochondria. In parallel, Survivin expression inhibits the respiratory complex-I, thereby preventing reactive oxygen species accumulation and, as a consequence, FOXO3-induced apoptosis. The loss of energy production via oxidative phosphorylation is compensated by increased glycolysis in Survivin-overexpressing NB tumor cells. Glycolysis inhibitors neutralize the antiapoptotic effect of Survivin and sensitize high-stage NB to DNA-damaging drugs. This suggests that glycolysis inhibitors target an 'archilles heel' of Survivin-overexpressing NB and may be highly useful as chemosensitizers in the treatment of high-stage NB.

It is shown for the first time that FOXO3/FKHRL1 induces caspase-8 expression via the ATM-CREB pathway independent of caspase-8 gene methylation status. Induction of caspase-8 by the DNA-methylation inhibitor 5-azadC also depends on FOXO3, suggesting that 5-azadC triggers gene expression via the FOXO3-ATM-CREB pathway., Forkhead box O (FOXO) transcription factors control diverse cellular functions, such as cell death, metabolism, and longevity. We analyzed FOXO3/FKHRL1 expression and subcellular localization in tumor sections of neuroblastoma patients and observed a correlation between nuclear FOXO3 and high caspase-8 expression. In neuroblastoma caspase-8 is frequently silenced by DNA methylation. Conditional FOXO3 activated caspase-8 gene expression but did not change the DNA-methylation pattern of regulatory sequences in the caspase-8 gene. Instead, FOXO3 induced phosphorylation of its binding partner ATM and of the ATM downstream target cAMP-responsive element binding protein (CREB), which was critical for FOXO3-mediated caspase-8 expression. Caspase-8 levels above a critical threshold sensitized neuroblastoma cells to tumor necrosis factor–related apoptosis-inducing ligand–induced cell death. The DNA-demethylating drug 5-Aza-2-deoxycytidine (5-azadC) induced rapid nuclear accumulation of FOXO3, ATM-dependent CREB phosphorylation, and caspase-8 expression in a FOXO3-dependent manner. This indicates that 5-azadC activates the FOXO3-ATM-CREB signaling pathway, which contributes to caspase-8 expression. The combined data suggest that FOXO3 is activated by 5-azadC treatment and triggers expression of caspase-8 in caspase-8–negative neuroblastoma, which may have important implication for metastasis, therapy, and death resistance of this childhood malignancy.

FOXO transcription factors induce apoptosis and regulate cellular production of reactive oxygen species (ROS). To identify the sequence of molecular events underlying FOXO3 (FKHRL1)-induced apoptosis, we studied the regulation and function of FOXO3 by expressing an ECFP-tagged FOXO3 or a 4OH-tamoxifen (4OHT)-inducible FOXO3–ERtm fusion protein in SH-EP and STA-NB15 neuronal cells. After knockdown of FOXO3 or expression of a dominant-negative FOXO3 mutant we observed that etoposide- and doxorubicin-induced elevation of cellular ROS depends on FOXO3 activation and induction of its transcriptional target BCL2L11 (Bim). Activation of FOXO3 on its own induced two sequential ROS waves as measured by reduced MitoTrackerRed in live cell microscopy. Induction of Bim by FOXO3 is essential for this phenomenon because Bim knockdown or ectopic expression of BCL2L1 (BclxL) prevented FOXO3-mediated overproduction of ROS and apoptosis. Tetracycline-controlled expression of Bim impaired mitochondrial respiration and caused ROS production, suggesting that FOXO3 induces uncoupling of mitochondrial respiration through Bim. FOXO3 also activated a ROS rescue pathway by inducing the peroxiredoxin SESN3 (Sestrin3), which is responsible for the biphasic ROS accumulation. Knockdown of SESN3 caused an increase of FOXO3-induced ROS and accelerated apoptosis. The combined data clearly demonstrate that FOXO3 activates overproduction of ROS as a consequence of Bim-dependent impairment of mitochondrial respiration in neuronal cells, which leads to apoptosis.

After undergoing several rounds of divisions normal human fibroblasts enter a terminally non-dividing state referred to as cellular or replicative senescence. We cloned MORF4 (mortality factor on human chromosome 4), as a cellular senescence inducing gene that caused immortal cells assigned to complementation group B for indefinite division to stop dividing. To facilitate analyses of this gene, which is toxic to cells at low levels, we obtained stable clones of HeLa cells expressing a tetracycline-induced MORF4 construct that could be induced by doxycycline in a dose-dependent manner. MORF4 induction resulted in reduced colony formation after 14 days of culture, as previously observed. We determined that MORF4 protein was unstable and that addition of the proteasome inhibitor MG132 resulted in the accumulation of the protein. Following removal of MG132 the protein was rapidly degraded. Subcellular fractionation following MG132 treatment demonstrated that the protein accumulates primarily in the cytoplasm with some amounts present in the nucleus. It is therefore possible that MORF4 protein, which escapes degradation in the cytoplasm, is transported to the nucleus where it is functional. The results suggest that levels of MORF4 in cells must be tightly controlled and one mechanism involves stability of the protein.

Loss of CDKN2A/p16INK4A in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia. This is also reflected by the frequent loss of the INK4A locus in acute lymphoblastic T-cell leukemia. T-cell acute lymphoblastic leukemia cells designed to conditionally express p16INK4A arrest in the G0/G1 phase of the cell cycle and show increased sensitivity to glucocorticoid- and tumor necrosis factor receptor superfamily 6-induced apoptosis. To investigate the underlying molecular mechanism for increased death sensitivity, we interfered with specific steps of apoptosis signaling by expression of anti-apoptotic proteins. We found that alterations in cell death susceptibility resulted from changes in the composition of pro- and anti-apoptotic BCL2 proteins, i.e. repression of MCL1, BCL2, and PMAIP1/Noxa and the induction of pro-apoptotic BBC3/Puma. Interference with Puma induction by short hairpin RNA technology or retroviral expression of MCL1 or BCL2 significantly reduced both glucocorticoid- and FAS-induced cell death in p16INK4A-reconstituted leukemia cells. These results suggest that Puma, in concert with MCL1 and BCL2 repression, critically mediates p16INK4A-induced death sensitization and that in human T-cell leukemia the deletion of p16INK4A confers apoptosis resistance by shifting the balance of pro- and anti-apoptotic BCL2 proteins toward apoptosis protection.

The phosphatidylinositol 3-kinase (PI3K)–protein kinase B (PKB) pathway regulates survival and chemotherapy resistance of neuronal cells, and its deregulation in neuroblastoma (NB) tumors predicts an adverse clinical outcome. Here, we show that inhibition of PI3K-PKB signaling in human NB cells induces nuclear translocation of FOXO3/FKHRL1, represses the prosurvival protein BIRC5/Survivin, and sensitizes to DNA-damaging agents. To specifically address whether FKHRL1 contributes to Survivin regulation, we introduced a 4-hydroxy-tamoxifen-regulated FKHRL1(A3)ERtm allele into NB cells. Conditional FKHRL1 activation repressed Survivin transcription and protein expression. Transgenic Survivin exerted a significant antiapoptotic effect and prevented the accumulation of Bim and Bax at mitochondria, the loss of mitochondrial membrane potential as well as the release of cytochrome c during FKHRL1-induced apoptosis. In concordance, Survivin knockdown by retroviral short hairpin RNA technology accelerated FKHRL1-induced apoptosis. Low-dose activation of FKHRL1 sensitized to the DNA-damaging agents doxorubicin and etoposide, whereas the overexpression of Survivin diminished FKHRL1 sensitization to these drugs. These results suggest that repression of Survivin by FKHRL1 facilitates FKHRL1-induced apoptosis and sensitizes to cell death induced by DNA-damaging agents, which supports the central role of PI3K-PKB-FKHRL1 signaling in drug resistance of human NB.

Tumor cells express the glycolytic regulator pyruvate kinase subtype M2 (M2-PK), which can occur in a tetrameric form with high affinity to its substrate phosphoenolpyruvate (PEP) and a dimeric form with a low PEP affinity. The transition between both conformations contributes to the control of glycolysis and is important for tumor cell proliferation and survival. Here we targeted M2-PK by synthetic peptide aptamers, which specifically bind to M2-PK and shift the isoenzyme into its low affinity dimeric conformation. The aptamer-induced dimerization and inactivation of M2-PK led to a significant decrease in the PK mass-action ratio as well as ATP:ADP ratio in the target cells. Furthermore, the expression of M2-PK-binding peptide aptamers moderately reduced the growth of immortalized NIH3T3 cell populations by decelerating cell proliferation, but without affecting apoptotic cell death. Moreover, the M2-PK-binding peptide aptamers also reduced the proliferation rate of human U-2 OS osteosarcoma cells. In the present study, we developed the first specific inhibitors of the pyruvate kinase isoenzyme type M2 and present evidence that these inhibitors moderately decelerate tumor cell proliferation.

The metabolic shift from oxidative phosphorylation to glycolysis as a hallmark of highly aggressive cancer was postulated by Otto Warburg in the 1920s. We identified baculoviral IAP repeat-containing 5 (BIRC5, also known as survivin) as a key player in mitochondrial metabolism and our recent findings suggest glycolysis inhibitors as powerful agents to overcome the antiapoptotic function of survivin in neuroblastoma.

Protein kinase-B (PKB) and its target, the forkhead transcription factor like 1 (FKHRL1)/FoxO3a, have been suggested as regulators of neurotrophin-mediated cell survival in neuronal cells. We analyzed human neuroblastoma cells and found that FKHRL1 was phosphorylated, suggesting its inactivation. To study FKHRL1 function, we infected SH-EP and NB15 cells with a 4OH-tamoxifen-regulated FKHRL1(A3)ER(tm) transgene. Activation of FKHRL1 promoted cytochrome-c release and caspase-dependent apoptosis. FKHRL1 induced TRAIL and the BH3-only proteins Noxa and Bim, implicating both extrinsic and intrinsic death pathways. However, expression of dnFADD did not inhibit FKHRL1-induced cell death, whereas Bcl2 protected against apoptosis. This excluded the death-receptor pathway and suggested that cell death decision is regulated by Bcl2-rheostat. Importantly, RNAi knockdown of Noxa or Bim decreased apoptosis, indicating that Noxa and Bim cooperate to mediate FKHRL1-induced cell death. We conclude that Noxa and Bim establish a connection between FKHRL1 and mitochondria, and that both BH3-only proteins are critically involved in FKHRL1-induced apoptosis in neuroblastoma.

Autosomal recessive polycystic kidney disease (ARPKD) is the most common pediatric renal cystic disease with liver involvement. The vast majority of patients with ARPKD carry mutations in the recently characterized PKHD1 gene on chromosome 6p12. A Turkish female demonstrated rapid growth of both kidneys after delivery. Accelerated growth of both kidneys and increasing respiratory distress necessitated right-sided nephrectomy at the age of three months. Because of persistent dyspnea and ongoing growth of the remaining kidney, the second kidney also had to be removed one month later. Biopsies taken from the kidney and the liver confirmed the diagnosis of ARPKD histologically. Renal ultrasound of the patient's consanguineous parents and her older brother showed normal results. PKHD1 mutation analysis yielded a novel homozygous missense mutation (c.1116C >G, F372L) in exon 14, coding for an Ig-like domain (TIG), possibly involved in the increased growth of the kidneys. Peritoneal dialysis was performed for 12 months. The patient had successful transplantation at the age of 15 months and is doing well with actual immunosuppression with cyclosporine, mycophenolate mofetil, and prednisolone. In conclusion, the present case clearly demonstrates the favorable outcome of a child with severe ARPKD after bilateral nephrectomy, pre-emptive dialysis, and successful transplantation.

Natural products are still an untapped source of promising lead compounds for the generation of antineoplastic drugs. Here, we investigated for the first time the antiproliferative and apoptotic effects of highly purified oxindole alkaloids, namely isopteropodine (A1), pteropodine (A2), isomitraphylline (A3), uncarine F (A4) and mitraphylline (A5) obtained from Uncaria tomentosa, a South American Rubiaceae, on human lymphoblastic leukaemia T cells (CCRF-CEM-C7H2). Four of the five tested alkaloids inhibited proliferation of acute lymphoblastic leukaemia cells. Furthermore, the antiproliferative effect of the most potent alkaloids pteropodine (A2) and uncarine F (A4) correlated with induction of apoptosis. After 48 h, 100 micromol/l A2 or A4 increased apoptotic cells by 57%. CEM-C7H2 sublines with tetracycline-regulated expression of bcl-2, p16ink4A or constitutively expressing the cowpox virus protein crm-A were used for further studies of the apoptosis-inducing properties of these alkaloids. Neither overexpression of bcl-2 or crm-A nor cell-cycle arrest in G0/G1 phase by tetracycline-regulated expression of p16INK4A could prevent alkaloid-induced apoptosis. Our results show the strong apoptotic effects of pteropodine and uncarine F on acute leukaemic lymphoblasts and recommend the alkaloids for further studies in xenograft models.