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The cross-regulation of metabolism and trafficking is not well understood for the vital sphingolipids and cholesterol constituents of cellular compartments. While reports are starting to surface on how sphingolipids like sphingomyelin (SM) dysregulate cholesterol levels in different cellular compartments (Jiang et al., 2022), limited research is available on the mechanisms driving the relationship between sphingolipids and cholesterol homeostasis, or its biological implications. Previously, we have identified sphingolipid metabolism as a unique vulnerability for IDH1 mut gliomas via a rational drug design. Herein, we show how modulating sphingolipid levels affects cholesterol homeostasis in brain tumors. However, we unexpectedly discovered for the first time that C17 sphingosine and NDMS addition to cancer cells alters cholesterol homeostasis by impacting its cellular synthesis, uptake, and efflux leading to a net decrease in cholesterol levels and inducing apoptosis. Our results reflect a reverse correlation between the levels of sphingosines, NDMS, and unesterified, free cholesterol in the cells. We show that increasing sphingosine and NDMS (a sphingosine analog) levels alter not only the trafficking of cholesterol between membranes but also the efflux and synthesis of cholesterol. We also demonstrate that despite the effort to remove free cholesterol by ABCA1-mediated efflux or by suppressing machinery for the influx (LDLR) and biosynthetic pathway (HMGCR), apoptosis is inevitable for IDH1 mut glioma cells. This is the first study that shows how altering sphingosine levels directly affects cholesterol homeostasis in cancer cells and can be used to manipulate this relationship to induce apoptosis in IDH1 mut gliomas.
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Oligodendroglioma is genetically defined as a tumor harboring isocitrate dehydrogenase 1 or 2 mutations (IDH1 mut /IDH2 mut ) and 1p/19q co-deletions. Previously, we reported that in IDH1 mut gliomas, D-2HG, the product of IDH1 mutant enzyme produces an increase in monounsaturated fatty acid levels that are incorporated into ceramides, tilting the S1P-to-ceramide rheostat toward apoptosis. Herein, we exploited this imbalance to further induce and IDH mut -specific glioma cell death. We report for the first time that the inhibition of acid ceramidase (AC) induces apoptosis and provides a benefit in mice survival in IDH1 mut oligodendroglioma. We demonstrated an IDH1 mut -specific cytotoxicity of SABRAC, an irreversible inhibitor of AC, in patient-derived oligodendroglioma cells. Exploring the mechanism of action of this drug, we found that SABRAC activates both extrinsic and intrinsic apoptosis in an ER stress-independent manner, pointing to a direct action of AC-related ceramides in mitochondria permeability. The activation of apoptosis detected under SABRAC treatment was associated with up to 30-fold increase in some ceramide levels and its derivatives from the salvage pathway. We propose that this novel enzyme, AC, has the potential to increase survival in oligodendroglioma with IDH1 mut and should be considered in the future.
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BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissue slides are routinely used in cancer diagnosis, clinical decision-making, and stored in biobanks, but their utilization in Raman spectroscopy-based studies has been limited due to the background coming from embedding media. METHODS: Spontaneous Raman spectroscopy was used for molecular fingerprinting of FFPE tissue from 46 patient samples with known methylation subtypes. Spectra were used to construct tumor/non-tumor, IDH1WT/IDH1mut, and methylation-subtype classifiers. Support vector machine and random forest were used to identify the most discriminatory Raman frequencies. Stimulated Raman spectroscopy was used to validate the frequencies identified. Mass spectrometry of glioma cell lines and TCGA were used to validate the biological findings. RESULTS: Here we develop APOLLO (rAman-based PathOLogy of maLignant glioma) - a computational workflow that predicts different subtypes of glioma from spontaneous Raman spectra of FFPE tissue slides. Our novel APOLLO platform distinguishes tumors from nontumor tissue and identifies novel Raman peaks corresponding to DNA and proteins that are more intense in the tumor. APOLLO differentiates isocitrate dehydrogenase 1 mutant (IDH1mut) from wildtype (IDH1WT) tumors and identifies cholesterol ester levels to be highly abundant in IDHmut glioma. Moreover, APOLLO achieves high discriminative power between finer, clinically relevant glioma methylation subtypes, distinguishing between the CpG island hypermethylated phenotype (G-CIMP)-high and G-CIMP-low molecular phenotypes within the IDH1mut types. CONCLUSIONS: Our results demonstrate the potential of label-free Raman spectroscopy to classify glioma subtypes from FFPE slides and to extract meaningful biological information thus opening the door for future applications on these archived tissues in other cancers.
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Cancer cell metabolism is often deregulated as a result of adaption to meeting energy and biosynthesis demands of rapid growth or direct mutation of key metabolic enzymes. Better understanding of such deregulation can provide new insights on targetable vulnerabilities, but is complicated by the difficulty in probing cell metabolism at different levels of resolution and under different experimental conditions. We construct computational models of glucose and glutamine metabolism with focus on the effect of IDH1/2-mutations in cancer using a combination of experimental metabolic flux data and patient-derived gene expression data. Our models demonstrate the potential of computational exploration to reveal biologic behavior: they show that an exogenously-mutated IDH1 experimental model utilizes glutamine as an alternative carbon source for lactate production under hypoxia, but does not fully-recapitulate the patient phenotype under normoxia. We also demonstrate the utility of using gene expression data as a proxy for relative differences in metabolic activity. We use the approach of probabilistic model checking and the freely-available Probabilistic Symbolic Model Checker to construct and reason about model behavior.
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Glutamina , Neoplasias , Humanos , Glutamina/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Modelos Estadísticos , Hipoxia de la Célula , Glucosa/metabolismoRESUMEN
Astrocytomas are the most common subtype of brain tumors and no curative treatment exist. Longitudinal assessment of patients, usually via Magnetic Resonance Imaging (MRI), is crucial since tumor progression may occur earlier than clinical progression. MRI usually provides a means for monitoring the disease, but it only informs about the structural changes of the tumor, while molecular changes can occur as a treatment response without any MRI-visible change. Radiotherapy (RT) is routinely performed following surgery as part of the standard of care in astrocytomas, that can also include chemotherapy involving temozolomide. Monitoring the response to RT is a key factor for the management of patients. Herein, we provide plasma and tissue metabolic biomarkers of treatment response in a mouse model of astrocytoma that was subjected to radiotherapy. Plasma metabolic profiles acquired over time by Liquid Chromatography Mass Spectrometry (LC/MS) were subjected to multivariate empirical Bayes time-series analysis (MEBA) and Receiver Operating Characteristic (ROC) assessment including Random Forest as the classification strategy. These analyses revealed a variation of the plasma metabolome in those mice that underwent radiotherapy compared to controls; specifically, fumarate was the best discriminatory feature. Additionally, Nuclear Magnetic Resonance (NMR)-based 13C-tracing experiments were performed at end-point utilizing [U-13C]-Glutamine to investigate its fate in the tumor and contralateral tissues. Irradiated mice displayed lower levels of glycolytic metabolites (e.g. phosphoenolpyruvate) in tumor tissue, and a higher flux of glutamine towards succinate was observed in the radiation cohort. The plasma biomarkers provided herein could be validated in the clinic, thereby improving the assessment of brain tumor patients throughout radiotherapy. Moreover, the metabolic rewiring associated to radiotherapy in tumor tissue could lead to potential metabolic imaging approaches for monitoring treatment using blood draws.
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Characterizing metabolism in cancer is crucial for understanding tumor biology and for developing potential therapies. Although most metabolic investigations analyze averaged metabolite levels from all cell compartments, subcellular metabolomics can provide more detailed insight into the biochemical processes associated with the disease. Methodological limitations have historically prevented the wider application of subcellular metabolomics in cancer research. Recently, however, ways to distinguish and identify metabolic pathways within organelles have been developed, including state-of-the-art methods to monitor metabolism in situ (such as mass spectrometry-based imaging, Raman spectroscopy and fluorescence microscopy), to isolate key organelles via new approaches and to use tailored isotope-tracing strategies. Herein, we examine the advantages and limitations of these developments and look to the future of this field of research.
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Metabolómica , Neoplasias , Diagnóstico por Imagen , Humanos , Espectrometría de Masas , Redes y Vías Metabólicas , Metabolómica/métodosRESUMEN
Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. However, little is known about the mechanisms that make cancer cells require certain nutrients from the microenvironment. Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13 C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to support glutathione synthesis through the transsulfuration pathway, which allows methionine to be converted to cysteine in cysteine/cystine-deprived conditions. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, although this increase did not attain statistical significance, with concomitant reductions in plasma glutathione and cysteine levels. At the end point, however, tumors displayed the ability to synthesize glutathione, even though higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolite levels in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.
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Cisteína , Glioma , Animales , Proliferación Celular , Cisteína/metabolismo , Cistina/metabolismo , Glutatión/metabolismo , Humanos , Ratones , Microambiente TumoralRESUMEN
Research in fundamental cell biology and pathology could be revolutionized by developing the capacity for quantitative molecular analysis of subcellular structures. To that end, we introduce the Ramanomics platform, based on confocal Raman microspectrometry coupled to a biomolecular component analysis algorithm, which together enable us to molecularly profile single organelles in a live-cell environment. This emerging omics approach categorizes the entire molecular makeup of a sample into about a dozen of general classes and subclasses of biomolecules and quantifies their amounts in submicrometer volumes. A major contribution of our study is an attempt to bridge Raman spectrometry with big-data analysis in order to identify complex patterns of biomolecules in a single cellular organelle and leverage discovery of disease biomarkers. Our data reveal significant variations in organellar composition between different cell lines. We also demonstrate the merits of Ramanomics for identifying diseased cells by using prostate cancer as an example. We report large-scale molecular transformations in the mitochondria, Golgi apparatus, and endoplasmic reticulum that accompany the development of prostate cancer. Based on these findings, we propose that Ramanomics datasets in distinct organelles constitute signatures of cellular metabolism in healthy and diseased states.
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Aparato de Golgi , Orgánulos , Biomarcadores/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Mitocondrias , Orgánulos/metabolismo , Espectrometría RamanRESUMEN
Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.
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Isocitrato Deshidrogenasa/genética , Mutación/genética , Orgánulos/metabolismo , Fosfolípidos/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Glioblastoma/patología , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Modelos Biológicos , Oligodendroglioma/patología , Estearoil-CoA Desaturasa/metabolismoRESUMEN
In addition to providing integrity to cellular structure, the various classes of lipids participate in a multitude of functions including secondary messengers, receptor stimulation, lymphocyte trafficking, inflammation, angiogenesis, cell migration, proliferation, necrosis and apoptosis, thus highlighting the importance of understanding their role in the tumor phenotype. In the context of IDH1mut glioma, investigations focused on metabolic alterations involving lipidomics' present potential to uncover novel vulnerabilities. Herein, a detailed lipidomic analysis of the sphingolipid metabolism was conducted in patient-derived IDH1mut glioma cell lines, as well as model systems, with the of identifying points of metabolic vulnerability. We probed the effect of decreasing D-2HG levels on the sphingolipid pathway, by treating these cell lines with an IDH1mut inhibitor, AGI5198. The results revealed that N,N-dimethylsphingosine (NDMS), sphingosine C17 and sphinganine C18 were significantly downregulated, while sphingosine-1-phosphate (S1P) was significantly upregulated in glioma cultures following suppression of IDH1mut activity. We exploited the pathway using a small-scale, rational drug screen and identified a combination that was lethal to IDHmut cells. Our work revealed that further addition of N,N-dimethylsphingosine in combination with sphingosine C17 triggered a dose-dependent biostatic and apoptotic response in a panel of IDH1mut glioma cell lines specifically, while it had little effect on the IDHWT cells probed here. To our knowledge, this is the first study that shows how altering the sphingolipid pathway in IDH1mut gliomas elucidates susceptibility that can arrest proliferation and initiate subsequent cellular death.
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BACKGROUND: Targeting glutamine metabolism in cancer has become an increasingly vibrant area of research. Mutant IDH1 (IDH1 mut ) gliomas are considered good candidates for targeting this pathway because of the contribution of glutamine to their newly acquired function: synthesis of 2-hydroxyglutarate (2HG). METHODS: We have employed a combination of 13C tracers including glutamine and glucose for investigating the metabolism of patient-derived IDH1 mut glioma cell lines through NMR and LC/MS. Additionally, genetic loss-of-function (in vitro and in vivo) approaches were performed to unravel the adaptability of these cell lines to the inhibition of glutaminase activity. RESULTS: We report the adaptability of IDH1 mut cells' metabolism to the inhibition of glutamine/glutamate pathway. The glutaminase inhibitor CB839 generated a decrease in the production of the downstream metabolites of glutamate, including those involved in the TCA cycle and 2HG. However, this effect on metabolism was not extended to viability; rather, our patient-derived IDH1 mut cell lines display a metabolic plasticity that allows them to overcome glutaminase inhibition. CONCLUSIONS: Major metabolic adaptations involved pathways that can generate glutamate by using alternative substrates from glutamine, such as alanine or aspartate. Indeed, asparagine synthetase was upregulated both in vivo and in vitro revealing a new potential therapeutic target for a combinatory approach with CB839 against IDH1 mut gliomas.
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Understanding the metabolic reprogramming of aggressive brain tumors has potential applications for therapeutics as well as imaging biomarkers. However, little is known about the nutrient requirements of isocitrate dehydrogenase 1 (IDH1) mutant gliomas. The IDH1 mutation involves the acquisition of a neomorphic enzymatic activity which generates D-2-hydroxyglutarate from α-ketoglutarate. In order to gain insight into the metabolism of these malignant brain tumors, we conducted metabolic profiling of the orthotopic tumor and the contralateral regions for the mouse model of IDH1 mutant glioma; as well as to examine the utilization of glucose and glutamine in supplying major metabolic pathways such as glycolysis and tricarboxylic acid (TCA). We also revealed that the main substrate of 2-hydroxyglutarate is glutamine in this model, and how this re-routing impairs its utilization in the TCA. Our 13C tracing analysis, along with hyperpolarized magnetic resonance experiments, revealed an active glycolytic pathway similar in both regions (tumor and contralateral) of the brain. Therefore, we describe the reprogramming of the central carbon metabolism associated with the IDH1 mutation in a genetically engineered mouse model which reflects the tumor biology encountered in glioma patients.
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BACKGROUND: Early detection of increased aggressiveness of brain tumors is a major challenge in the field of neuro-oncology because of the inability of traditional imaging to uncover it. Isocitrate dehydrogenase (IDH)-mutant gliomas represent an ideal model system to study the molecular mechanisms associated with tumorigenicity because they appear indolent and non-glycolytic initially, but eventually a subset progresses toward secondary glioblastoma with a Warburg-like phenotype. The mechanisms and molecular features associated with this transformation are poorly understood. METHODS: We employed model systems for IDH1 mutant (IDH1mut) gliomas with different growth and proliferation rates in vivo and in vitro. We described the metabolome, transcriptome, and epigenome of these models in order to understand the link between their metabolism and the tumor biology. To verify whether this metabolic reprogramming occurs in the clinic, we analyzed data from The Cancer Genome Atlas. RESULTS: We reveal that the aggressive glioma models have lost DNA methylation in the promoters of glycolytic enzymes, especially lactate dehydrogenase A (LDHA), and have increased mRNA and metabolite levels compared with the indolent model. We find that the acquisition of the high glycolytic phenotype occurs at the glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP)-high molecular subtype in patients and is associated with the worst outcome. CONCLUSION: We propose very early monitoring of lactate levels as a biomarker of metabolic reprogramming and tumor aggressiveness.
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Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/genética , Metilación de ADN , Glioma/genética , Guanina , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Mutación , FenotipoRESUMEN
Detailed studies of lipids in biological systems, including their role in cellular structure, metabolism, and disease development, comprise an increasingly prominent discipline called lipidomics. However, the conventional lipidomics tools, such as mass spectrometry, cannot investigate lipidomes until they are extracted, and thus they cannot be used for probing the lipid distribution nor for studying in live cells. Furthermore, conventional techniques rely on the lipid extraction from relatively large samples, which averages the data across the cellular populations and masks essential cell-to-cell variations. Further advancement of the discipline of lipidomics critically depends on the capability of high-resolution lipid profiling in live cells and, potentially, in single organelles. Here we report a micro-Raman assay designed for single-organelle lipidomics. We demonstrate how Raman microscopy can be used to measure the local intracellular biochemical composition and lipidome hallmarks-lipid concentration and unsaturation level, cis/trans isomer ratio, sphingolipids and cholesterol levels in live cells-with a sub-micrometer resolution, which is sufficient for profiling of subcellular structures. These lipidome data were generated by a newly developed biomolecular component analysis software, which provides a shared platform for data analysis among different research groups. We outline a robust, reliable, and user-friendly protocol for quantitative analysis of lipid profiles in subcellular structures. This method expands the capabilities of Raman-based lipidomics toward the analysis of single organelles within either live or fixed cells, thus allowing an unprecedented measure of organellar lipid heterogeneity and opening new quantitative ways to study the phenotypic variability in normal and diseased cells.
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Lipidómica/métodos , Microscopía Óptica no Lineal/métodos , Orgánulos/química , Análisis de la Célula Individual/métodos , Espectrometría Raman/métodos , Metabolismo de los Lípidos , Lípidos/análisis , Orgánulos/metabolismo , Programas InformáticosRESUMEN
Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Glucólisis/efectos de los fármacos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Sarcoma de Ewing/patología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Sarcoma de Ewing/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Raman microspectroscopy is a rapidly developing technique, which has an unparalleled potential for in situ proteomics, lipidomics, and metabolomics, due to its remarkable capability to analyze the molecular composition of live cells and single cellular organelles. However, the scope of Raman spectroscopy for bio-applications is limited by a lack of software tools for express-analysis of biomolecular composition based on Raman spectra. In this study, we have developed the first software toolbox for immediate analysis of intracellular Raman spectra using a powerful biomolecular component analysis (BCA) algorithm. Our software could be easily integrated with commercial Raman spectroscopy instrumentation, and serve for precise analysis of molecular content in major cellular organelles, including nucleoli, endoplasmic reticulum, Golgi apparatus, and mitochondria of either live or fixed cells. The proposed software may be applied in broad directions of cell science, and serve for further advancement and standardization of Raman spectroscopy.
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Algoritmos , Espectrometría Raman/métodos , Microscopía , Orgánulos , Programas InformáticosRESUMEN
Background: Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods: Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results: PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions: Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.
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Biomarcadores de Tumor/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cordoma/tratamiento farmacológico , Cordoma/patología , Piperazinas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Animales , Apoptosis , Movimiento Celular , Proliferación Celular , Cordoma/enzimología , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas, fibrosarcoma, and other cancers leads to a novel metabolite, D-2-hydroxyglutarate, which is proposed to cause tumorigenesis. The production of this metabolite also causes vulnerabilities in cellular metabolism, such as lowering NADPH levels. To exploit this vulnerability, we treated glioma and fibrosarcoma cells that harbor an IDH1 mutation with an inhibitor of nicotinamide adenine dinucleotide (NADâº) salvage pathway, FK866, and observed decreased viability in these cells. To understand the mechanism of action by which the inhibitor FK866 works, we used Raman imaging microscopy and identified that proteins and lipids are decreased upon treatment with the drug. Raman imaging showed a different distribution of lipids throughout the cell in the presence of the drug compared with the untreated cells. We employed nuclear magnetic resonance NMR spectroscopy and mass spectrometry to identify the classes of lipids altered. Our combined analyses point to a decrease in cell division due to loss of lipid content that contributes to membrane formation in the in vitro setting. However, the FK866 drug did not have the same potency in vivo. The use of Raman imaging microscopy indicated an opposite trend of lipid distribution in the tissue collected from treated versus untreated mice when compared with the cells. These results demonstrate the role of Raman imaging microscopy to identify and quantify metabolic changes in cancer cells and tissue.
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Acrilamidas/farmacología , Antineoplásicos/farmacología , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Piperidinas/farmacología , Espectrometría Raman , Animales , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones SCID , Neoplasias Experimentales/tratamiento farmacológico , Células Tumorales CultivadasRESUMEN
Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients.Experimental Design: To investigate TG02, an agent with known penetration of the blood-brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays.Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue.Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124-37. ©2017 AACR.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral/trasplante , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Ratones , Ratones Endogámicos C57BL , Temozolomida/farmacología , Temozolomida/uso terapéuticoRESUMEN
Aniline-peptide (FLDQV, FLDQVC, Dansyl-FLDQV, Dansyl-FLDQVC, and FLDQV-AMC) mixtures underwent oxidative chemical and electrochemical polymerization in excess of aniline. The products of the chemical polymerization were low molecular weight polymers containing more than 70% peptide. Electrochemically polymerized species polyaniline-FLDQV (PANI-FLDQV) consisted mainly of polyaniline units containing about 10% peptide. The solubility of the latter in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) was similar to the camphorsulfonic acid (CSA) doped emeraldine base (PANI-CSA) solubility, however the weight composition of the electrospun fibers produced from the two polymers was significantly different. 2D 1H-13C HSQC analyses were employed to analyze the binding between the aniline and peptide moieties. Binding of peptide to polyaniline is reflected by the appearance of extra cross-peaks which display line broadening between the free polyaniline and the free pentapeptide. Peptides may be chemically bonded to the polymer molecules, but they may also act as doping agents to the nitrogen atoms via hydrogen bonding.