Prophylaxis with enoxaparin and antithrombin III in drug-induced coagulation alterations in childhood leukemia: a retrospective experience of 20 years.

BACKGROUND: Thromboembolic complications are well known in the treatment of childhood acute lymphoblastic leukemia. Over the years it has not been possible to reach a consensus on a possible prophylaxis of thromboembolic events during intensive therapy. Only the administration of enoxaparin was able to achieve evidence in the literature to date. METHODS: In this retrospective study, 173 childhood leukemia patients were treated over 20 years with a thromboembolic prophylaxis including enoxaparin and AT III during induction therapy with L-asparaginase and cortisone. RESULTS: We here report the effectiveness of administration of enoxaparin and AT III in childhood leukemia, showing a strikingly low prevalence of deep vein thrombosis (2.9%). Especially in adolescent patients, a particularly great need for AT III was demonstrated. CONCLUSIONS: We recommend thromboembolic prophylaxis with enoxaparin and AT III substitution during induction/reinduction therapy with L-asparaginase and glucocorticosteroids, especially from adolescence onwards.

C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.

BACKGROUND: FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells. METHODS: The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor. RESULTS: We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells. CONCLUSION: The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.

FOXO3-induced reactive oxygen species are regulated by BCL2L11 (Bim) and SESN3.

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.

Recommendations for Nutritional Supplementation in Pediatric Oncology: A Compilation of the Facts.

BACKGROUND: As one of the few modifiable risk factors, the importance of dietary patterns for both disease prevention and treatment outcome in pediatric oncology has gained increasing popularity. Malnutrition is associated with lower survival rates, tolerance to treatment, and quality of life. Yet, especially in children with malignancies, nutritional deterioration is common, and pediatric cancer patients often present with inadequate intake of micro- and macronutrients alike. Despite the reported widespread use of dietary supplements, few empirical data provide a basis for clinical recommendations, and evidence for their efficacy is inconsistent. Current literature lacks a systematic approach as to how and which supplements should be recommended for specific patients, types of cancer, or during specific treatments. The aim of this review is to highlight the role of the most frequently used nutrients in pediatric malignant diseases and to give a practical guide based on current scientific evidence. METHODS: A comprehensive literature search was conducted on PubMed through April 2023 to select meta-analyses, systematic reviews, observational studies, and individual randomized controlled trials (RCTs) of macro- and micronutrient supplementation in pediatric oncology. The search strategy included the following medical subject headings (MeSH) and keywords: "childhood cancer", "pediatric oncology", "nutritional status", "dietary supplements", "vitamins", "micronutrients", "calcium", "magnesium", "vitamin D", "zinc" "glutamine", "selen", and "omega-3 fatty acids". The reference lists of all relevant articles were screened to include potentially pertinent studies. RESULTS: The present review provides a comprehensive and updated overview of the latest evidence about the role of nutrition and diet in pediatric oncology, also focusing on different nutritional interventions available for the management of the disease. We summarize evidence about the importance of adequate nutrition in childhood cancer and the role of several micronutrients and critically interpret the findings. Possible effects and benefits of supplementation during chemotherapy are discussed, as are strategies for primary and secondary prevention. CONCLUSIONS: We here describe the obvious benefits of dietary supplementation for childhood cancer. Further large-scale clinical trials are required to verify the impacts of deficiencies and the possible benefits of supplementation and optimal dosages. (337 words).

Isolation and characterization of CD133+CD34+VEGFR-2+CD45- fetal endothelial cells from human term placenta.

The phenotypes and functions of endothelial cells (EC), a heterogeneous cell population, vary along the vascular tree and even in the same organ between different vessels. The placenta is an organ with abundant vessels. To enhance further knowledge concerning placenta derived EC, we develop a new method for isolation, purification and culture of these EC. Moreover, in order to investigate the peculiarity of placenta derived EC we compare their phenotypic and functional characteristics with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Freshly isolated placenta derived EC displayed an elongated shape with pale cytoplasm and showed the typical cobblestone pattern of EC but also a swirling pattern when confluent. FISH-analyses of the isolated EC from placentae of male fetus revealed an XY genotype strongly indicating their fetal origin. Characterisation of placenta derived fetal EC (fEC) underlined their blood vessel phenotype by the expression of vWF, Ulex europaeus lectin-1, HLA-class I molecules, CD31, CD34, CD36, CD51/61, CD54, CD62E, CD105, CD106, CD133, CD141, CD143, CD144, CD146, VEGFR-1, VEGFR-2, EN-4, PAL-E, BMA120, Tie-1, Tie-2 and α-Tubulin. In contrast to previous reports the expression of lymphatic markers, like VEGFR-3, LYVE-1, Prox-1 and Podoplanin was consistently negative. Haematopoietic surface markers like CD45 and CD14 were also always negative. Various functional tests (Dil-Ac-LDL uptake, Matrigel assay and TNF-α induced upregulation of CD62E and CD54) substantiated the endothelial nature of propagated fEC. At the ultrastructural level, fEC harboured numerous microvilli, micropinocytic vesicles at their basis, were rich in intermediate filaments and possessed typical Weibel - Palade bodies. In conclusion, the placenta is a plentiful source of fetal, microvascular, blood EC with an expression profile (CD34+, CD133+, VEGFR-2+, CD45-) suggestive of an endothelial progenitor phenotype.

Supportive methods for childhood acute lymphoblastic leukemia then and now: A compilation for clinical practice.

Survival of childhood acute lymphoblastic leukemia has significantly improved over the past decades. In the early years of chemotherapeutic development, improvement in survival rates could be attained only by increasing the cytostatic dose, also by modulation of the frequency and combination of chemotherapeutic agents associated with severe short- and long-time side-effects and toxicity in a developing child's organism. Years later, new treatment options have yielded promising results through targeted immune and molecular drugs, especially in relapsed and refractory leukemia, and are continuously added to conventional therapy or even replace first-line treatment. Compared to conventional strategies, these new therapies have different side-effects, requiring special supportive measures. Supportive treatment includes the prevention of serious acute and sometimes life-threatening events as well as managing therapy-related long-term side-effects and preemptive treatment of complications and is thus mandatory for successful oncological therapy. Inadequate supportive therapy is still one of the main causes of treatment failure, mortality, poor quality of life, and unsatisfactory long-term outcome in children with acute lymphoblastic leukemia. But nowadays it is a challenge to find a way through the flood of supportive recommendations and guidelines that are available in the literature. Furthermore, the development of new therapies for childhood leukemia has changed the range of supportive methods and must be observed in addition to conventional recommendations. This review aims to provide a clear and recent compilation of the most important supportive methods in the field of childhood leukemia, based on conventional regimes as well as the most promising new therapeutic approaches to date.

The anti-apoptotic protein BCL2L1/Bcl-xL is neutralized by pro-apoptotic PMAIP1/Noxa in neuroblastoma, thereby determining bortezomib sensitivity independent of prosurvival MCL1 expression.

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

Tackling resistance in chronic myeloid leukemia: Novel cell death modulators with improved efficacy.

The development of resistance poses a serious problem in the therapy of cancer due to the necessity of a multiple-drug and unlimited treatment of affected patients. In chronic myeloid leukemia (CML), the introduction of imatinib has revolutionized the therapy. The persistence of an untreatable cancer stem cell pool and other resistance-causing factors, however, also impede the cure of this malignancy. New therapeutic approaches are therefore essential to overcome current treatment drawbacks. In this regard, an intervention in the STAT5 signaling pathway can significantly improve drug response, as this central signaling node induces the formation of highly resistant CML cells. In the present study, we continued the design of efficient chemosensitizers derived from the partial PPARγ agonist telmisartan. The developed 2-carbonitriles or 2-carboxymethyl esters showed improved potency in sensitizing K562-resistant cells to imatinib treatment, even at concentrations, which are considered patient-relevant. At 5 µM, for instance, 2d sensitized the cells in such a manner that the resistance was fully overcome and the recovered efficacy of imatinib resulted in >76% cell death. Importantly, all compounds were non-cytotoxic per se. A transactivation experiment showed that only the carbonitriles are partial agonists of PPARγ, which does not seem to be involved in the mode of action. Yet, immunoassays revealed a suppression of the STAT5 phosphorylation status by co-application of the most active derivatives with imatinib. This mechanism consequently resulted in reduced cell proliferation and induction of cell death in resistant CML cells.

p16INK4A sensitizes human leukemia cells to FAS- and glucocorticoid-induced apoptosis via induction of BBC3/Puma and repression of MCL1 and BCL2.

Loss of CDKN2A/p16(INK4A) 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 p16(INK4A) arrest in the G(0)/G(1) 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 p16(INK4A)-reconstituted leukemia cells. These results suggest that Puma, in concert with MCL1 and BCL2 repression, critically mediates p16(INK4A)-induced death sensitization and that in human T-cell leukemia the deletion of p16(INK4A) confers apoptosis resistance by shifting the balance of pro- and anti-apoptotic BCL2 proteins toward apoptosis protection.

Toll-like receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors.

Toll-like receptor 9 (TLR9) activates the innate immune response when exposed to non-methylated CpG-DNA. TLR9 was recently shown to be expressed by cancer cells which have been previously characterized by global hypomethylation. We set out to examine the expression and molecular activity of TLR9 in breast and ovarian cancer cells. Firstly, we confirmed higher levels of hypomethylated DNA in the serum of patients with metastatic breast cancer (n = 18) versus age-matched tumor-free women (n = 18). In breast cancer cell lines and tissues, TLR9 mRNA expression was associated with estrogen-receptor (ER) status (n = 124, P = 0.005). Expression also correlated with increasing tumor grade in both breast (P = 0.03) and ovarian cancer specimens (n = 138, P = 0.04). Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) breast cancer tissues revealed higher TLR9 protein expression in hormone-receptor (HR)-negative specimens (n = 116, P < 0.001). Using an in vitro scratch assay, we observed that cell lines transfected to overexpress TLR9 demonstrated increased cellular migration when stimulated with CpG-DNA. When assessing the molecular activity of TLR9 in breast cancer, we found a strong positive correlation of nuclear factor-kappa B (NF-kappaB) activity with TLR9 mRNA expression (correlation coefficient r = 0.7, P < 0.001). Finally, immunofluorescence analysis of BT-20 and Hs578T breast cancer cell lines showed partial colocalizations of CpG-DNA with TLR9, which diminished when the cells were exposed to methylated CpG-DNA (mCpG-DNA) or control GpC-DNA. In summary we demonstrate that TLR9 expression is associated with poor differentiation in breast and ovarian cancer specimens, and that TLR9 overexpression and stimulation with hypomethylated DNA augments the migratory capacity of cancer cell lines.

Identification and development of non-cytotoxic cell death modulators: Impact of sartans and derivatives on PPARγ activation and on growth of imatinib-resistant chronic myelogenous leukemia cells.

4'-((2-Propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-carboxylic acid derived from telmisartan was identified as lead for the design of cell death modulators. In this study, we evaluated the efficacy of telmisartan itself and other sartans in combination with imatinib against K562-resistant cells. The findings were directly used to further optimize the lead structure. Telmisartan and candesartan cilexetil represented the most effective sartans, thus the influence of carboxyl/methyl carboxylate groups at positions 7 (compounds 6, 7) or 4 (compounds 12-14) at the benzimidazole core was studied. Additionally, according to the results of a former structure-activity study, telmisartan was transformed to the related amide (1). Telmisartan amide 1, as well as the esters 6 and 12 markedly sensitized the resistant CML cells to imatinib treatment. Correlation with their potency to activate PPARγ is not given. Candesartan cilexetil, telmisartan and 1 showed the profile of partial agonists at PPARγ with EC50 values of 4.2, 4.3 and 9.1 µM, respectively, while 6 and 12 caused only marginal intrinsic activation at 10 µM (Amax = 22% and 13%). However, the repression of the STAT5 phosphorylation relates with the possibility to sensitize K562-resistant CML cells to imatinib treatment. It is worth mentioning that all compounds were per se non-cytotoxic at relevant concentrations.

Overcoming imatinib resistance in chronic myelogenous leukemia cells using non-cytotoxic cell death modulators.

Recent studies examined the possibility to overcome imatinib resistance in chronic myeloid leukemia (CML) patients by combination therapy with peroxisome proliferator-activated receptor gamma (PPARγ) ligands. Pioglitazone, a full PPARγ agonist, improved the survival of patients by the gradual elimination of the residual CML stem cell pool. To evaluate the importance of the pharmacological profile of PPARγ agonists on the ability to circumvent resistance, the partial PPARγ agonist 4'-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-carboxylic acid, derived from telmisartan, and other related derivatives were investigated. The 4-substituted benzimidazole derivatives bearing a [1,1'-biphenyl]-2-carboxamide moiety sensitized K562-resistant cells to imatinib treatment. Especially the derivatives 18a-f, which did not activate PPARγ to more than 40% at 10 µM, retrieved the cytotoxicity of imatinib in these cells. The cell death modulating properties were higher than that of pioglitazone. It is of interest to note that all novel compounds were not cytotoxic neither on non-resistant nor on resistant cells. They exerted antitumor potency only in combination with imatinib.

Synthesis and Characterization of Telmisartan-Derived Cell Death Modulators to Circumvent Imatinib Resistance in Chronic Myeloid Leukemia.

New strategies to eradicate cancer stem cells in chronic myeloid leukemia (CML) include a combination of imatinib with peroxisome proliferator-activated receptor gamma (PPARγ) ligands. Recently, we identified the partial PPARγ agonist telmisartan as effective sensitizer of resistant K562 CML cells to imatinib treatment. Here, the importance of the heterocyclic core on the cell death-modulating effects of the telmisartan-derived lead 4'-((2-propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1'-biphenyl]-2-carboxylic acid (3 b) was investigated. Inspired by the pharmacodynamics of HYL-6d and the selective PPARγ ligand VSP-51, the benzimidazole was replaced by a carbazole or an indole core. The results indicate no correlation between PPARγ activation and sensitization of resistant CML cells to imatinib. The 2-COOH derivatives of the carbazoles or indoles achieved low activity at PPARγ, while the benzimidazoles showed 60-100 % activation. Among the 2-CO2 CH3 derivatives, only the ester of the lead (2 b) slightly activated PPARγ. Sensitizing effects were further observed for this non-cytotoxic 2 b (80 % cell death), and to a lesser extent for the lead 3 b or the 5-Br-substituted ester of the benzimidazoles (5 b).

A drug library screen identifies Carbenoxolone as novel FOXO inhibitor that overcomes FOXO3-mediated chemoprotection in high-stage neuroblastoma.

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.

Endothelial cells from cord blood CD133+CD34+ progenitors share phenotypic, functional and gene expression profile similarities with lymphatics.

The existence of endothelial progenitor cells (EPC) with high cell-cycle rate in human umbilical cord blood has been recently shown and represents a challenging strategy for therapeutic neovascularization. To enhance knowledge for future cellular therapy, we compared the phenotypic, functional and gene expression differences between EPC-derived cells generated from cord blood CD34(+) cells, and lymphatic and macrovascular endothelial cells (EC) isolated from human foreskins and umbilical veins, respectively. Under appropriate culture conditions, EPC developed into fully matured EC with expression of similar endothelial markers as lymphatic and macrovascular EC, including CD31, CD36, von Willebrand factor FVIII, CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-cadherin), Tie-1, Tie-2, VEGFR-1/Flt-1 and VEGFR-2/Flk-1. Few EPC-derived cells became positive for LYVE-1, indicating their origin from haematopoietic stem cells. However they lacked expression of other lymphatic cell-specific markers such as podoplanin and Prox-1. Functional tests demonstrated that the cobblestone EPC-derived cells up-regulated CD54 and CD62E expression in response to TNF-alpha, incorporated DiI-acetylated low-density liproprotein and formed cord- and tubular-like structures with capillary lumen in three-dimensional collagen culture--all characteristic features of the vascular endothelium. Structures compatible with Weibel-Palade bodies were also found by electron microscopy. Gene microarray profiling revealed that only a small percentage of genes investigated showed differential expression in EPC-derived cells and lymphatic EC. Among them were adhesion molecules, extracellular matrix proteins and cytokines. Our data point to the close lineage relationship of both types of vascular cells and support the theory of a venous origin of the lymphatic system.

Repaglinide Silences the FOXO3/Lumican Axis and Represses the Associated Metastatic Potential of Neuronal Cancer 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 "wound healing" 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.

A New Approach in Cancer Treatment: Discovery of Chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(III) Complexes as Ferroptosis Inducers.

Chlorido[N,N'-disalicylidene-1,2-phenylenediamine]iron(III) complexes generate lipid-based ROS and induce ferroptosis in leukemia and neuroblastoma cell lines. The extent of ferroptosis on the mode of action is regulated by simple modifications of the substituents at the 1,2-phenylenediamine moiety. In HL-60 cells, the unsubstituted lead exclusively caused ferroptosis. For instance, a 4-F substituent shifted the mode of action toward both ferroptosis and necroptosis, while the analogously chlorinated derivative exerted only necroptosis. Remarkably, cell-death in NB1 neuroblastoma cells was solely induced by ferroptosis, independent of the used substituents. The effects were higher than that of the therapeutically applied drug cisplatin. These data clearly demonstrate for the first time that not only iron ions but also iron salophene complexes are potent ferroptosis inducers, which can be optimized as antitumor agents.

Modulating FOXO3 transcriptional activity by small, DBD-binding molecules.

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.

A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes.

Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27(Kip1) affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27(Kip1) expression caused transient G(0)-G(1) accumulation without reduced viability, whereas high p27(Kip1) levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27(Kip1) as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells.

The tubulin inhibitor MG-2477 induces autophagy-regulated cell death, ROS accumulation and activation of FOXO3 in neuroblastoma.

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.