RESUMO
Prostate cancer (PCa) is the second most diagnosed malignancy and the fifth leading cause of cancer associated death in men worldwide. Dysregulation of cellular energetics has become a hallmark of cancer, evidenced by numerous connections between signaling pathways that include oncoproteins and key metabolic enzymes. We previously showed that heme oxygenase 1 (HO-1), a cellular homeostatic regulator counteracting oxidative and inflammatory damage, exhibits anti-tumoral activity in PCa cells, inhibiting cell proliferation, migration, tumor growth and angiogenesis. The aim of this study was to assess the role of HO-1 on the metabolic signature of PCa. After HO-1 pharmacological induction with hemin, PC3 and C4-2B cells exhibited a significantly impaired cellular metabolic rate, reflected by glucose uptake, ATP production, lactate dehydrogenase (LDH) activity and extracellular lactate levels. Further, we undertook a bioinformatics approach to assess the clinical significance of LDHA, LDHB and HMOX1 in PCa, identifying that high LDHA or low LDHB expression was associated with reduced relapse free survival (RFS). Interestingly, the shortest RFS was observed for PCa patients with low HMOX1 and high LDHA, while an improved prognosis was observed for those with high HMOX1 and LDHB. Thus, HO-1 induction causes a shift in the cellular metabolic profile of PCa, leading to a less aggressive phenotype of the disease.
RESUMO
RSUME (for RWD-domain-containing sumoylation enhancer), RWDD3 gene, was identified from a pituitary tumor cell with increased tumorigenic and angiogenic potential, and has higher expression in cerebellum, pituitary, heart, kidney, liver, pancreas, adrenal gland and prostate. RSUME is induced by cellular stress like hypoxia and heat shock, and is increased in pituitary tumors, in gliomas and in VHL tumors. Seven splicing forms have been described. Two of them correspond to non-coding RNAs and the other five possess an RWD domain in the N-terminus and differ in their C-terminal end. RSUME enhances SUMO conjugation by interacting with the SUMO conjugase Ubc9, increases Ubc9 thioester formation and therefore favors sumoylation of specific targets. RSUME increases IκB levels and stabilizes HIF-1α during hypoxia, leading to inhibition of NF-κB and increased HIF-1 transcriptional activity. RSUME inhibits pVHL function, thus suppressing HIF-1 and 2α ubiquitination and degradation. Disruption of the RWD domain structure of RSUME indicated that this domain is critical for RSUME action. The findings point to an important role of RSUME in the regulation and stability of specific targets, which are key regulatory mediators in cancer and inflammation.
Assuntos
Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Hipóxia Celular , Humanos , Inflamação/metabolismo , Inflamação/patologia , Neoplasias/patologiaRESUMO
Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O2, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O2 utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis.
Assuntos
Ciclo Celular , Mitocôndrias/fisiologia , Animais , Morte Celular , Proliferação de Células , Humanos , Oxirredução , Fosfotransferases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Hemorrhage (H) is associated with a left ventricular (LV) dysfunction. However, the diastolic function has not been studied in detail. The main goal was to assess the diastolic function both during and 120 min after bleeding, in the absence and in the presence of L-NAME. Also, the changes in mRNA and protein expression of nitric oxide synthase (NOS) isoforms were determined. New Zealand rabbits were divided into three groups: Sham group, H group (hemorrhage 20% blood volume), and H L-NAME group (hemorrhage treated with L-NAME). We evaluated systolic and diastolic ventricular functions in vivo and in vitro (Langendorff technique). Hemodynamic parameters and LV function were measured before, during, and at 120 min after bleeding. We analyzed the isovolumic relaxation using t ½ in vivo (closed chest). After that, hearts were excised and perfused in vitro to measure myocardial stiffness. Samples were frozen to measure NOS mRNA and protein expression. The t½ increased during bleeding and returned to basal values 120 min after bleeding. L-NAME blunted this effect. Data from the H group revealed a shift to the left in the LV end diastolic pressure-volume curve at 120 min after bleeding, which was blocked by L-NAME. iNOS and nNOS protein expression and mRNA levels increased at 120 min after the hemorrhage. Acute hemorrhage induces early and transient isovolumic relaxation impairment and an increase in myocardial stiffness 120 min after bleeding. L-NAME blunted the LV dysfunction, suggesting that NO modulates ventricular function through iNOS and nNOS isoforms.
Assuntos
Diástole , Choque Hemorrágico/fisiopatologia , Disfunção Ventricular Esquerda/tratamento farmacológico , Animais , Diástole/efeitos dos fármacos , Diástole/fisiologia , Coração/fisiopatologia , Hemorragia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Óxidos de Nitrogênio , Coelhos , Choque Hemorrágico/complicações , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/etiologiaRESUMO
The subcellular localization and physiological functions of biomolecules are closely related and thus it is crucial to precisely determine the distribution of different molecules inside the intracellular structures. This is frequently accomplished by fluorescence microscopy with well-characterized markers and posterior evaluation of the signal colocalization. Rigorous study of colocalization requires statistical analysis of the data, albeit yet no single technique has been established as a standard method. Indeed, the few methods currently available are only accurate in images with particular characteristics. Here, we introduce a new algorithm to automatically obtain the true colocalization between images that is suitable for a wide variety of biological situations. To proceed, the algorithm contemplates the individual contribution of each pixel's fluorescence intensity in a pair of images to the overall Pearsons correlation and Manders' overlap coefficients. The accuracy and reliability of the algorithm was validated on both simulated and real images that reflected the characteristics of a range of biological samples. We used this algorithm in combination with image restoration by deconvolution and time-lapse confocal microscopy to address the localization of MEK1 in the mitochondria of different cell lines. Appraising the previously described behavior of Akt1 corroborated the reliability of the combined use of these techniques. Together, the present work provides a novel statistical approach to accurately and reliably determine the colocalization in a variety of biological images.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Sistema de Sinalização das MAP Quinases/fisiologia , Mitocôndrias/metabolismo , Algoritmos , Animais , Biomarcadores , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Células NIH 3T3 , Plasmídeos/metabolismo , Fatores de TempoRESUMO
Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser(473) by mTORC2 and Thr(308) by PDK1. On these bases, we investigated the mechanistic connection of H(2)O(2) yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H(2)O(2) entails the entrance of cytosolic P-Akt1 Ser(473) to mitochondria, where it is further phosphorylated at Thr(308) by constitutive PDK1. Phosphorylation of Thr(308) in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H(2)O(2), Akt1-PDK1 association is disrupted and P-Akt1 Ser(473) accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H(2)O(2) effects on Akt1-PDK interaction depend on the selective oxidation of Cys(310) to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.
Assuntos
Mitocôndrias/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Ciclo Celular , Linhagem da Célula , Ácido Cisteico/química , Citosol/metabolismo , Peróxido de Hidrogênio/química , Camundongos , Modelos Biológicos , Células NIH 3T3 , Fosforilação , Ácidos Sulfênicos/químicaRESUMO
Phylogenetic studies had shown that evolution of mitochondria occurred in parallel with the maturation of kinases implicated in growth and final size of modern organisms. In the last years, different reports confirmed that MAPKs, Akt, PKA and PKC are present in mitochondria, particularly in the intermembrane space and inner membrane where they meet mitochondrial constitutive upstream activators. Although a priori phosphorylation is the apparent aim of translocation, new perspectives indicate that kinase activation depends on redox status as determined by the mitochondrial production of oxygen species. We observed that the degree of mitochondrial oxidation of ERK Cys(38) and Cys(214) discriminates the kinase to be phosphorylated and determines translocation to the nuclear compartment and proliferation, or accumulation in mitochondria and arrest. Otherwise, transcriptional gene regulation by Akt depends on Cys(60) and Cys(310) oxidation to sulfenic and sulfonic acids. It is concluded that the interactions between kinases and mitochondria control cell signaling pathways and participate in the modulation of cell proliferation and arrest, tissue protection, tumorigenesis and cancer progression.
Assuntos
Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Proteínas Quinases/fisiologia , Transdução de Sinais/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Humanos , Modelos Biológicos , Infarto do Miocárdio/metabolismo , Neoplasias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mitochondria are specialized organelles that control energy metabolism and also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or, conversely, promote cell arrest and programmed cell death by a limited number of oxidative or nitrative reactions. Nitric oxide (NO) regulates oxygen uptake by reversible inhibition of cytochrome oxidase and the production of superoxide anion from the mitochondrial electron transfer chain. In this sense, NO produced by mtNOS will set the oxygen uptake level and contribute to oxidation-reduction reaction (redox)-dependent cell signaling. Modulation of translocation and activation of neuronal nitric oxide synthase (mtNOS activity) under different physiologic or pathologic conditions represents an adaptive response properly modulated to adjust mitochondria to different cell challenges.
Assuntos
Metabolismo Energético , Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Óxido Nítrico Sintase/metabolismo , Estresse Fisiológico , Óxido Nítrico/metabolismoRESUMO
Mitochondria are major cellular sources of hydrogen peroxide (H(2)O(2)), the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2)O(2) concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2)O(2) due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs) orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2)O(2) yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2)O(2) increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2)O(2) arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei), the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs) facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2)O(2) level. Like this, high H(2)O(2) or directed mutation of redox-sensitive ERK2 Cys(214) impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to increase H(2)O(2) yield should disrupt synchronized MAPK oxidations and the regulation of cell cycle leading cells to remain in a proliferating phenotype.
Assuntos
Mitocôndrias/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/patologia , Animais , Catálise , Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Camundongos , Neoplasias/enzimologia , Oxirredução , Fenótipo , Fosforilação , Transporte Proteico , Transdução de SinaisRESUMO
In the last years, nitric oxide synthases (NOS) have been localized in mitochondria. At this site, NO yield directly regulates the activity of cytochrome oxidase, O(2) uptake and the production of reactive oxygen species. Recent studies showed that translocated neuronal nitric oxide synthase (nNOS) is posttranslationally modified including phosphorylation at Ser 1412 (in mice) and myristoylation in an internal residue. Different studies confirm that modified nNOS alpha is the main modulable isoform in mitochondria. Modulation of mtNOS was observed in different situations, like adaptation to reduced O(2) availability and hypoxia, adaptation to low environmental temperature, and processes linked to life and death by effects on kinases and transcription factors. We present here evidence about the role of mtNOS in the analyzed conditions.
Assuntos
Mitocôndrias/enzimologia , Óxido Nítrico Sintase Tipo I/fisiologia , Adaptação Fisiológica , Animais , Encéfalo/embriologia , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Fígado/embriologia , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Camundongos , Plasticidade Neuronal , Oxigênio/metabolismo , Tri-Iodotironina/fisiologiaRESUMO
Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-alpha (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3',5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Hipotireoidismo/patologia , Fígado/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Ciclina D1/metabolismo , Citosol/metabolismo , Elétrons , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/metabolismo , Hipotireoidismo/metabolismo , Immunoblotting , Imunoprecipitação , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Microscopia Imunoeletrônica , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/metabolismo , Modelos Químicos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/metabolismo , Oxidantes/metabolismo , Oxigênio/metabolismo , Ácido Peroxinitroso/química , Fenótipo , Isoformas de Proteínas , Transporte Proteico , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Frações Subcelulares/metabolismo , Hormônios Tireóideos/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mitochondrial nitric oxide synthase (mtNOS) is a fine regulator of oxygen uptake and reactive oxygen species that eventually modulates the activity of regulatory proteins and cell cycle progression. From this perspective, we examined liver mtNOS modulation and mitochondrial redox changes in developing rats from embryonic days 17-19 and postnatal day 2 (proliferating hepatocyte phenotype) through postnatal days 15-90 (quiescent phenotype). mtNOS expression and activity were almost undetectable in fetal liver, and progressively increased after birth by tenfold up to adult stage. NO-dependent mitochondrial hydrogen peroxide (H(2)O(2)) production and Mn-superoxide dismutase followed the developmental modulation of mtNOS and contributed to parallel variations of cytosolic H(2)O(2) concentration ([H(2)O(2)](ss)) and cell fluorescence. mtNOS-dependent [H(2)O(2)](ss) was a good predictor of extracellular signal-regulated kinase (ERK)/p38 activity ratio, cyclin D1, and tissue proliferation. At low 10(-11)-10(-12) M [H(2)O(2)](ss), proliferating phenotypes had high cyclin D1 and phospho-ERK1/2 and low phospho-p38 mitogen-activated protein kinase, while at 10(-9) M [H(2)O(2)](ss), quiescent phenotypes had the opposite pattern. Accordingly, leading postnatal day 2-isolated hepatocytes to embryo or adult redox conditions with H(2)O(2) or NO-H(2)O(2) scavengers, or with ERK inhibitor U0126, p38 inhibitor SB202190 or p38 activator anisomycin resulted in correlative changes of ERK/p38 activity ratio, cyclin D1 expression, and [(3)H] thymidine incorporation in the cells. Accordingly, p38 inhibitor SB202190 or N-acetyl-cysteine prevented H(2)O(2) inhibitory effects on proliferation. In conclusion, the results suggest that a synchronized increase of mtNOS and derived H(2)O(2) operate on hepatocyte signaling pathways to support the liver developmental transition from proliferation to quiescence.