RESUMEN
Mitochondria have been recognized as the energy (in the form of ATP)-producing cell organelles, required for cell viability, survival and normal cell function [...].
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Mitocondrias , Orgánulos , Mitocondrias/metabolismo , Supervivencia Celular , Dinámicas Mitocondriales , Metabolismo EnergéticoRESUMEN
BACKGROUND: The opioid agonist D,L-methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. In preclinical tumor-models D,L-methadone shows apoptotic and chemo-sensitizing effects and was therefore hyped as an off-label "anticancer" drug without substantiation from clinical trials. Its effects in ovarian cancer (OC) are completely unexplored. METHODS: We analyzed OPRM1-mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 170 OC tissue 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 three 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 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 also detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect.
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Antineoplásicos , Neoplasias Ováricas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Femenino , Humanos , Metadona/farmacología , Metadona/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Factor A de Crecimiento Endotelial VascularRESUMEN
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.
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Mitocondrias Cardíacas/fisiología , Músculo Esquelético/fisiología , Miocitos Cardíacos/fisiología , Animales , Respiración de la Célula/fisiología , Metabolismo Energético/fisiología , HumanosRESUMEN
Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired ß-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 ß-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.
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Caprilatos/farmacología , Fibroblastos/efectos de los fármacos , Heptanoatos/farmacología , Errores Innatos del Metabolismo Lipídico/metabolismo , Lipidómica/métodos , Proteómica/métodos , Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Cardiolipinas/metabolismo , Línea Celular , Femenino , Fibroblastos/metabolismo , Genotipo , Humanos , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/patología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Proteoma/metabolismo , Esfingolípidos/metabolismoRESUMEN
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.
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Neoplasias Encefálicas/genética , Factores de Transcripción Forkhead/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas/genética , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Sitios de Unión/genética , Neoplasias Encefálicas/patología , Catalasa/metabolismo , Etopósido/farmacología , Proteína Forkhead Box O3 , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Peróxido de Hidrógeno/toxicidad , Péptidos y Proteínas de Señalización Intracelular , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuroblastoma/patología , Peroxisomas/efectos de los fármacos , Peroxisomas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , beta Catenina/metabolismoRESUMEN
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.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Factores de Transcripción Forkhead/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Doxorrubicina/farmacología , Etopósido/farmacología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/genética , Humanos , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Neuroblastoma/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes de Fusión/metabolismo , Tetraciclina/farmacologíaRESUMEN
Besides their main function for energy production in form of ATP in processes of oxidative phosphorylation (OxPhos), mitochondria perform many other important cellular functions and participate in various physiological processes that are congregated. For example, mitochondria are considered to be one of the main sources of reactive oxygen species (ROS) and therefore they actively participate in the regulation of cellular redox and ROS signaling. These organelles also play a crucial role in Ca2+ signaling and homeostasis. The mitochondrial OxPhos and their cellular functions are strongly cell/tissue specific and can be heterogeneous even within the same cell, due to the existence of mitochondrial subpopulations with distinct functional and structural properties. However, the interplay between different functions of mitochondria is not fully understood. The mitochondrial functions may change as a response to the changes in the cellular metabolism (signaling in). On the other hand, several factors and feedback signals from mitochondria may influence the entire cell physiology (signaling out). Numerous interactions between mitochondria and the rest of cell, various cytoskeletal proteins, endoplasmic reticulum (ER) and other cellular elements have been demonstrated, and these interactions could actively participate in the regulation of mitochondrial and cellular metabolism. This review highlights the important role of the interplay between mitochondrial and entire cell physiology, including signaling from and to mitochondria.
RESUMEN
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.
RESUMEN
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.
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Células Madre Mesenquimatosas , Neuroblastoma , Preescolar , Células Endoteliales , Humanos , Hidrogeles/química , Hidrogeles/farmacología , Neuroblastoma/tratamiento farmacológico , Medicina de Precisión , Impresión Tridimensional , Ingeniería de Tejidos , Microambiente TumoralRESUMEN
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.
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Antineoplásicos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Ácidos Borónicos/metabolismo , Neuroblastoma/metabolismo , Inhibidores de Proteasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazinas/metabolismo , Proteína bcl-X/metabolismo , Animales , Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Ácidos Borónicos/uso terapéutico , Bortezomib , Caspasas/metabolismo , Línea Celular Tumoral , Niño , Resistencia a Antineoplásicos , Activación Enzimática , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Inhibidores de Proteasas/uso terapéutico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Pirazinas/uso terapéutico , Receptores de Muerte Celular/metabolismo , Transducción de Señal/fisiología , Transgenes , Proteína bcl-X/genéticaRESUMEN
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.
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Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Benzoatos/farmacología , Núcleo Celular/metabolismo , Proliferación Celular , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Citoplasma/metabolismo , Doxiciclina/farmacología , Inhibidores Enzimáticos/farmacología , Furanos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Células HeLa , Humanos , Leupeptinas/farmacología , Oligopéptidos/farmacología , Péptidos/genética , Inhibidores de Proteasoma , Pirazoles/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Transfección , Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
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.
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Bioimpresión , Animales , Bioimpresión/métodos , Células Cultivadas , Humanos , Mamíferos , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
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.
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Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Neoplasias del Colon/metabolismo , Desoxiglucosa/farmacología , Metabolismo Energético , Mitocondrias/fisiología , Fosforilación Oxidativa , Proliferación Celular , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Glucólisis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Mitocondrias/efectos de los fármacos , Dinámicas MitocondrialesRESUMEN
Long-chain fatty acid oxidation disorders (lc-FAOD) are a group of diseases affecting the degradation of long-chain fatty acids. In order to investigate the disease specific alterations of the cellular lipidome, we performed undirected lipidomics in fibroblasts from patients with carnitine palmitoyltransferase II, very long-chain acyl-CoA dehydrogenase, and long-chain 3-hydroxyacyl-CoA dehydrogenase. We demonstrate a deep remodeling of mitochondrial cardiolipins. The aberrant phosphatidylcholine/phosphatidylethanolamine ratio and the increased content of plasmalogens and of lysophospholipids support the theory of an inflammatory phenotype in lc-FAOD. Moreover, we describe increased ratios of sphingomyelin/ceramide and sphingomyelin/hexosylceramide in LCHAD deficiency which may contribute to the neuropathic phenotype of LCHADD/mitochondrial trifunctional protein deficiency.
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Ácidos Grasos/metabolismo , Fibroblastos/enzimología , Errores Innatos del Metabolismo Lipídico/enzimología , Lipidómica , Metaboloma , Piel/enzimología , Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Cardiolipinas/metabolismo , Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/genética , Estudios de Casos y Controles , Células Cultivadas , Ceramidas/metabolismo , Femenino , Humanos , Errores Innatos del Metabolismo Lipídico/genética , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/deficiencia , 3-Hidroxiacil-CoA Deshidrogenasa de Cadena Larga/genética , Masculino , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Oxidación-Reducción , Esfingolípidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
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.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Regulación hacia Abajo , Proteína Ligando Fas/metabolismo , Leucemia/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación Leucémica de la Expresión Génica , Glucocorticoides/metabolismo , Humanos , Leucemia/genética , Leucemia/fisiopatología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Elucidation of the mitochondrial regulatory mechanisms for the understanding of muscle bioenergetics and the role of mitochondria is a fundamental problem in cellular physiology and pathophysiology. The cytoskeleton (microtubules, intermediate filaments, microfilaments) plays a central role in the maintenance of mitochondrial shape, location, and motility. In addition, numerous interactions between cytoskeletal proteins and mitochondria can actively participate in the regulation of mitochondrial respiration and oxidative phosphorylation. In cardiac and skeletal muscles, mitochondrial positions are tightly fixed, providing their regular arrangement and numerous interactions with other cellular structures such as sarcoplasmic reticulum and cytoskeleton. This can involve association of cytoskeletal proteins with voltage-dependent anion channel (VDAC), thereby, governing the permeability of the outer mitochondrial membrane (OMM) to metabolites, and regulating cell energy metabolism. Cardiomyocytes and myocardial fibers demonstrate regular arrangement of tubulin beta-II isoform entirely co-localized with mitochondria, in contrast to other isoforms of tubulin. This observation suggests the participation of tubulin beta-II in the regulation of OMM permeability through interaction with VDAC. The OMM permeability is also regulated by the specific isoform of cytolinker protein plectin. This review summarizes and discusses previous studies on the role of cytoskeletal proteins in the regulation of energy metabolism and mitochondrial function, adenosine triphosphate (ATP) production, and energy transfer.
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Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Miocardio/citología , Metabolismo Energético , Humanos , Mitocondrias/metabolismo , Modelos BiológicosRESUMEN
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.
Asunto(s)
Carbenoxolona/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Proteína Forkhead Box O3/antagonistas & inhibidores , Proteína Forkhead Box O3/metabolismo , Neuroblastoma/patología , Bibliotecas de Moléculas Pequeñas , Carbenoxolona/química , Muerte Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Humanos , Peso Molecular , Estadificación de Neoplasias , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Encefálicas/metabolismo , Dipéptidos/farmacología , Indoles/farmacología , Dinámicas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Neuroblastoma/metabolismo , Tiazoles/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Proteínas Reguladoras de la Apoptosis/química , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Dipéptidos/administración & dosificación , Dipéptidos/uso terapéutico , Resistencia a Antineoplásicos , Células HEK293 , Humanos , Indoles/administración & dosificación , Indoles/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Mitocondriales/química , Neuroblastoma/tratamiento farmacológico , Survivin/metabolismo , Tiazoles/administración & dosificación , Tiazoles/uso terapéutico , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
FOXO transcription factors regulate cellular homeostasis, longevity and response to stress. FOXO1 (also known as FKHR) is a key regulator of hepatic glucose production and lipid metabolism, and its specific inhibition may have beneficial effects on diabetic hyperglycemia by reducing hepatic glucose production. Moreover, all FOXO proteins are considered potential drug targets for drug resistance prevention in cancer therapy. However, the development of specific FOXO inhibitors requires a detailed understanding of structural differences between individual FOXO DNA-binding domains. The high-resolution structure of the DNA-binding domain of FOXO1 reported in this study and its comparison with structures of other FOXO proteins revealed differences in both their conformation and flexibility. These differences are encoded by variations in protein sequences and account for the distinct functions of FOXO proteins. In particular, the positions of the helices H1, H2 and H3, whose interface form the hydrophobic core of the Forkhead domain, and the interactions between hydrophobic residues located on the interface between the N-terminal segment, the H2-H3 loop, and the recognition helix H3 differ among apo FOXO1, FOXO3 and FOXO4 proteins. Therefore, the availability of apo structures of DNA-binding domains of all three major FOXO proteins will support the development of FOXO-type-specific inhibitors.
Asunto(s)
Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Animales , Proteína Forkhead Box O1/química , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/química , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Factores de Transcripción Forkhead/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Análisis de Secuencia de ProteínaRESUMEN
Mitochondria play a critical role in maintaining cellular function by ATP production. They are also a source of reactive oxygen species (ROS) and proapoptotic factors. The role of mitochondria has been established in many aspects of cell physiology/pathophysiology, including cell signaling. Mitochondria may deteriorate under various pathological conditions, including ischemia-reperfusion (IR) injury. Mitochondrial injury can be one of the main causes for cardiac and other tissue injuries by energy stress and overproduction of toxic reactive oxygen species, leading to oxidative stress, elevated calcium and apoptotic and necrotic cell death. However, the interplay among these processes in normal and pathological conditions is still poorly understood. Mitochondria play a critical role in cardiac IR injury, where they are directly involved in several pathophysiological mechanisms. We also discuss the role of mitochondria in the context of mitochondrial dynamics, specializations and heterogeneity. Also, we wanted to stress the existence of morphologically and functionally different mitochondrial subpopulations in the heart that may have different sensitivities to diseases and IR injury. Therefore, various cardioprotective interventions that modulate mitochondrial stability, dynamics and turnover, including various pharmacologic agents, specific mitochondrial antioxidants and uncouplers, and ischemic preconditioning can be considered as the main strategies to protect mitochondrial and cardiovascular function and thus enhance longevity.