RESUMEN
Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.
Asunto(s)
Neoplasias , Nucleótidos de Purina , Purinas , Animales , Ratones , Purinas/metabolismo , Purinas/biosíntesis , Neoplasias/metabolismo , Neoplasias/patología , Nucleótidos de Purina/metabolismo , Humanos , Inosina/metabolismo , Hipoxantina/metabolismo , Ratones Endogámicos C57BL , Adenina/metabolismo , Línea Celular Tumoral , FemeninoRESUMEN
Patients with comorbidities of obesity and diabetes are recognized to be at high risk of breast cancer development and face worse breast cancer outcomes. Though several reports showed the reinforced link between obesity, diabetes, and prediabetes with breast cancer, the underlying molecular mechanisms are still unknown. The present study aimed to investigate the underlying molecular link between increased risks of breast cancer due to coincident diabetes or obesity using a spontaneous obese rat model with impaired glucose tolerance (WNIN/GR-Ob rat). A single dose of solubilized DMBA suspension (40 mg/kg body weight) was orally administered to the animals at the age of 60 days to induce breast tumors. The tumor incidence, latency period, tumor frequency, and tumor volume were measured. Histology, immunohistochemistry, and immunoblotting were performed to evaluate the tumor morphology and expression levels of signal molecules. The development of mammary tumors in GR-Ob rats was characterized by early onset and shorter latency periods compared to control lean rats. While 62% of obese rats developed breast tumors, tumor development in lean rats was only 21%. Overexpression of ER, PR, Ki67, and p53 markers was observed in tumor tissues of obese rats in comparison with lean rats. The levels of the hallmarks of cell proliferation and angiogenesis involved in IGF-1/PI3K/Akt/GSK3ß/ß-catenin signaling pathway molecules were upregulated in obese rat breast tumors compared to lean rats. Furthermore, obesity with prediabetes is associated with changes in IGF-1 signaling and acts on PI3K/Akt/GSK3ß/ß-catenin signaling, which results in rapid cell proliferation and development of breast tumors in obese rats than the lean rats. These results indicate that tumor onset and development were faster in spontaneous obese rat models with impaired glucose tolerance than in their lean counterparts.
Asunto(s)
Intolerancia a la Glucosa , Neoplasias , Estado Prediabético , Ratas , Animales , Intolerancia a la Glucosa/complicaciones , Glucógeno Sintasa Quinasa 3 beta , Factor I del Crecimiento Similar a la Insulina , beta Catenina , Estado Prediabético/complicaciones , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Obesidad/metabolismo , Neoplasias/complicacionesRESUMEN
Myopathies secondary to mitochondrial electron transport chain (ETC) dysfunction can result in devastating disease. While the consequences of ETC defects have been extensively studied in culture, little in vivo data are available. Using a mouse model of severe, early-onset mitochondrial myopathy, we characterized the proteomic, transcriptomic, and metabolic characteristics of disease progression. Unexpectedly, ETC dysfunction in muscle results in reduced expression of glycolytic enzymes in our animal model and patient muscle biopsies. The decrease in glycolysis was mediated by loss of constitutive Hif1α signaling, down-regulation of the purine nucleotide cycle enzyme AMPD1, and activation of AMPK. In vivo isotope tracing experiments indicated that myopathic muscle relies on lactate import to supply central carbon metabolites. Inhibition of lactate import reduced steady-state levels of tricarboxylic acid cycle intermediates and compromised the life span of myopathic mice. These data indicate an unexpected mode of metabolic reprogramming in severe mitochondrial myopathy that regulates disease progression.
RESUMEN
NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.
Asunto(s)
Lisina , NAD , Acetilación , Dominio Catalítico , Humanos , Lisina/metabolismo , Proteínas Mitocondriales/metabolismo , NAD/metabolismo , NADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Prolina/metabolismoRESUMEN
Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.
Asunto(s)
Nucleótidos de Purina , Serina , Carbono , Movimiento Celular , Purinas , Serina/metabolismoRESUMEN
Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.
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Neoplasias de la Mama/metabolismo , Epóxido Hidrolasas/metabolismo , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinogénesis , Proliferación Celular/fisiología , Transformación Celular Neoplásica , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Epóxido Hidrolasas/genética , Compuestos Epoxi/metabolismo , Ácidos Grasos Insaturados/metabolismo , Femenino , Eliminación de Gen , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismoRESUMEN
Nicotinamide adenine dinucleotide phosphate (NADP+) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP+, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.
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Proliferación Celular , Mitocondrias/metabolismo , NADP/metabolismo , Prolina/biosíntesis , Animales , Ciclo Celular/genética , Humanos , Ratones , Ratones Noqueados , Consumo de Oxígeno , Páncreas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Especies Reactivas de Oxígeno/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The molecular elements that govern cellular transformation and tumorigenic competence remain poorly understood. Metabolic reprogramming has emerged as a hallmark of malignant transformation. Recently in Cell Metabolism, Zhang et al. showed that an increase of cellular antioxidant capacity and nucleotide availability is sufficient to induce oncogenic transformation and tumorigenesis.
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Antioxidantes , Nucleótidos , Carcinogénesis , Transformación Celular Neoplásica , HumanosRESUMEN
Arachidonic acid epoxides generated by cytochrome P450 (CYP) enzymes have been linked to increased tumor growth and metastasis, largely on the basis of overexpression studies and the application of exogenous epoxides. Here we studied tumor growth and metastasis in Cyp2c44-/- mice crossed onto the polyoma middle T oncogene (PyMT) background. The resulting PyMT2c44 mice developed more primary tumors earlier than PyMT mice, with increased lymph and lung metastasis. Primary tumors from Cyp2c44-deficient mice contained higher numbers of tumor-associated macrophages, as well as more lymphatic endothelial cells than tumors from PyMT mice. While epoxide and diol levels were comparable in tumors from both genotypes, prostaglandin (PG) levels were higher in the PyMTΔ2c44 tumors. This could be accounted for by the finding that Cyp2c44 metabolized the PG precursor, PGH2 to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), thus effectively reducing levels of effector PGs (including PGE2). Next, proteomic analyses revealed an up-regulation of WD repeating domain FYVE1 (WDFY1) in tumors from PyMTΔ2c44 mice, a phenomenon that was reproduced in Cyp2c44-deficient macrophages as well as by PGE2 Mechanistically, WDFY1 was involved in Toll-like receptor signaling, and its down-regulation in human monocytes attenuated the LPS-induced phosphorylation of IFN regulatory factor 3 and nuclear factor-κB. Taken together, our results indicate that Cyp2c44 protects against tumor growth and metastasis by preventing the synthesis of PGE2 The latter eicosanoid influenced macrophages at least in part by enhancing Toll-like receptor signaling via the up-regulation of WDFY1.
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Neoplasias de la Mama/metabolismo , Familia 2 del Citocromo P450/metabolismo , Linfangiogénesis/fisiología , Prostaglandinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Familia 2 del Citocromo P450/genética , Modelos Animales de Enfermedad , Células Endoteliales/patología , Ácidos Grasos Insaturados/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Linfangiogénesis/genética , Macrófagos , Ratones , Ratones Noqueados , Monocitos , Procesos Neoplásicos , Proteómica , Transducción de Señal , Receptores Toll-Like , Regulación hacia ArribaRESUMEN
BACKGROUND: Blocking vascular endothelial growth factor (VEGF) mediated tumor angiogenesis by phytochemicals has emerged as an attractive strategy for cancer prevention and therapy. METHODS: We investigated the anti-angiogenic effects of ellagic acid in a hamster model of oral oncogenesis by examining the transcript and protein expression of hypoxia-inducible factor-1alpha (HIF-1α), VEGF, VEGFR2, and the members of the PI3K/Akt and MAPK signaling cascades. Molecular docking studies and cell culture experiments with the endothelial cell line ECV304 were performed to delineate the mechanism by which ellagic acid regulates VEGF signaling. RESULTS: We found that ellagic acid significantly inhibits HIF-1α-induced VEGF/VEGFR2 signalling in the hamster buccal pouch by abrogating PI3K/Akt and MAPK signaling via downregulation of PI3K, PDK-1, p-Akt(ser473), mTOR, p-ERK, and p-JNK. Ellagic acid was also found to reduce the expression of histone deacetylases that could inhibit neovascularization. Analysis of the mechanism revealed that ellagic acid inhibits hypoxia-induced angiogenesis via suppression of HDAC-6 in ECV304 cells. Furthermore, knockdown of endogenous HDAC6 via small interfering RNA abrogated hypoxia-induced expression of HIF-1α and VEGF and blocked Akt activation. Molecular docking studies confirmed interaction of ellagic acid with upstream kinases that regulate angiogenic signaling. CONCLUSIONS: Taken together, these findings demonstrate that the anti-angiogenic activity of ellagic acid may be mediated by abrogation of hypoxia driven PI3K/Akt/mTOR, MAPK and VEGF/VEGFR2 signaling pathways involving suppression of HDAC6 and HIF-1α responses. GENERAL SIGNIFICANCE: Ellagic acid offers promise as a lead compound for anticancer therapeutics by virtue of its ability to inhibit key oncogenic signaling cascades and HDACs.
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Inhibidores de la Angiogénesis/farmacología , Carcinogénesis/efectos de los fármacos , Ácido Elágico/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Oncogénica v-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinogénesis/inducido químicamente , Carcinogénesis/metabolismo , Carcinogénesis/patología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Mejilla/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Histona Desacetilasas/metabolismo , Masculino , Mesocricetus , Simulación del Acoplamiento Molecular , Neovascularización Patológica/prevención & control , Transducción de SeñalRESUMEN
Plant-derived polyphenolic compounds have beneficial health effects. In the present study, we determined the ability of ellagic acid (EA) to prevent platelet-derived growth factor-BB (PDGF-BB)-induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs). We also determined the ability of EA to prevent atherosclerosis in streptozotocin-induced diabetic rats. Proliferation of cells was measured via Alamar Blue assay and through propidium iodide-based cell cycle analysis in flow cytometer. Reactive oxygen species (ROS) were measured via 2',7'-dichlorofluorescin diacetate and Amplex red methods. Expression of proliferation markers and activation of kinases were assessed by immunoblot analysis. Cotreatment of primary cultures of RASMCs with 25 µmol/L of EA significantly reduced PDGF-BB (20 ng/ml)-induced proliferation by blocking S-phase entry. EA effectively blocked PDGF receptor-ß (PDGFR-ß) tyrosine phosphorylation, generation of intracellular ROS and downstream activation of extracellular signal-regulated kinase 1/2. It also blocked PDGF-BB-induced expression of cyclin D1. Computational molecular docking of EA with the PDGFR-ß-PDGF-BB complex revealed two putative inhibitor binding sites which showed similar binding energies with the known PDGFR-ß inhibitor AG1295. In diabetic rats, supplementation of diet with 2% EA significantly blocked diabetes-induced medial thickness, and lipid and collagen deposition in the arch of aorta. These were assessed through haematoxylin and eosin, Oil Red O and Masson's trichome staining, respectively. EA treatment also blocked cyclin D1 expression in medial smooth muscle cells in experimental animals. Thus, EA is effective in reducing atherosclerotic process by blocking proliferation of vascular smooth muscle cells.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Ácido Elágico/farmacología , Placa Aterosclerótica/prevención & control , Proteínas Proto-Oncogénicas c-sis/farmacología , Animales , Becaplermina , Sitios de Unión , Ciclina D1/biosíntesis , Masculino , Simulación del Acoplamiento Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis.