RESUMO
F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ionóforos de Próton/farmacologiaRESUMO
BACKGROUND: The epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis and is also associated with drug resistance. Thus, controlling EMT status is a research of interest to conquer the malignant tumors. MATERIALS AND METHODS: A drug repositioning analysis of transcriptomic data from a public cell line database identified monensin, a widely used in veterinary medicine, as a candidate EMT inhibitor that suppresses the conversion of the EMT phenotype. Using TGF-ß-induced EMT cell line models, the effects of monensin on the EMT status and EMT-mediated drug resistance were assessed. RESULTS: TGF-ß treatment induced EMT in non-small cell lung cancer (NSCLC) cell lines and the EGFR-mutant NSCLC cell lines with TGF-ß-induced EMT acquired resistance to EGFR-tyrosine kinase inhibitor. The addition of monensin effectively suppressed the TGF-ß-induced-EMT conversion, and restored the growth inhibition and the induction of apoptosis by the EGFR-tyrosine kinase inhibitor. CONCLUSION: Our data suggested that combined therapy with monensin might be a useful strategy for preventing EMT-mediated acquired drug resistance.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Monensin/farmacologia , Ionóforos de Próton/farmacologia , Antifúngicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Sobrevivência Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Receptores ErbB/antagonistas & inibidores , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/farmacologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Dinâmica Mitocondrial , Mitofagia , Músculo Esquelético/patologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Ativação Enzimática , Células HeLa , Humanos , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Ionóforos de Próton/farmacologia , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
BACKGROUND: Cellular metabolic changes that accompany malignant transformation have been heralded as hallmark features of cancer. However, metabolic signatures between neoplasms can be unique, allowing for distinctions in malignancy, invasion and chemoresistance between cancer types and subtypes. Mitochondria are central metabolic mediators, as cellular bioenergetics veers from oxidative phosphorylation to glycolysis. Herein, we evaluate the role of mitochondria in maintenance of cellular metabolism, proliferation, and survival in the adult granulosa tumor cell line, KGN, as well as three epithelial ovarian cancer cell lines to determine distinctions in specific features. RESULTS: Notably, KGN cells were susceptible to TRAIL- and cisplatin-induced death following pretreatment with the metabolic inhibitor FCCP, but not oligomycin A. Collapse of mitochondrial membrane potential was found concomitant with cell death via apoptosis, independent from extrinsic canonical apoptotic routes. Rather, treatment with FCCP resulted in elevated cytochrome c release from mitochondria and decreased responsiveness to BIRC5. Following knockdown of BIRC5, mitochondrial membrane depolarization further sensitized KGN cells to induction of apoptosis via TRAIL. CONCLUSIONS: These results indicate an essential role, distinct from metabolism, for mitochondrial membrane potential in KGN cells to sense and respond to external mediators of apoptotic induction.
Assuntos
Tumor de Células da Granulosa/fisiopatologia , Potencial da Membrana Mitocondrial , Neoplasias Ovarianas/fisiopatologia , Survivina/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Morte Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Humanos , Oligomicinas/farmacologia , Ionóforos de Próton/farmacologia , Survivina/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
There are considerable challenges in directly targeting the mutant p53 protein, given the large heterogeneity of p53 mutations in the clinic. An alternative approach is to exploit the altered fitness of cells imposed by loss-of-wild-type p53. Here we identify niclosamide through a HTS screen for compounds selectively killing p53-deficient cells. Niclosamide impairs the growth of p53-deficient cells and of p53 mutant patient-derived ovarian xenografts. Metabolome profiling reveals that niclosamide induces mitochondrial uncoupling, which renders mutant p53 cells susceptible to mitochondrial-dependent apoptosis through preferential accumulation of arachidonic acid (AA), and represents a first-in-class inhibitor of p53 mutant tumors. Wild-type p53 evades the cytotoxicity by promoting the transcriptional induction of two key lipid oxygenation genes, ALOX5 and ALOX12B, which catalyzes the dioxygenation and breakdown of AA. Therefore, we propose a new paradigm for targeting cancers defective in the p53 pathway, by exploiting their vulnerability to niclosamide-induced mitochondrial uncoupling.
Assuntos
Mitocôndrias/efeitos dos fármacos , Niclosamida/uso terapêutico , Ionóforos de Próton/uso terapêutico , Proteína Supressora de Tumor p53/deficiência , Animais , Apoptose , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico , Cálcio/metabolismo , Técnicas de Cocultura , Células HCT116 , Humanos , Metabolismo dos Lipídeos , Metaboloma/efeitos dos fármacos , Camundongos , Niclosamida/farmacologia , Ionóforos de Próton/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Reactive oxygen species (ROS) and mitophagy are profoundly implicated in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease (PD). Several studies have suggested that ROS are not involved in mitochondrial translocation of Parkin which primes mitochondria for autophagic elimination. However, whether ROS play a role in the execution of mitophagy is unknown. In the present study, we show that carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment induced both mitochondrial depolarization and generation of ROS that were needed for the mitophagy process. Cells failed to proceed to complete mitophagy if CCCP treatment was discontinued even after recruitment of Parkin and autophagy machinery to mitochondria. Notably, treatment of pro-oxidant was able to replace CCCP treatment to take mitophagy forward, while it alone was insufficient to induce translocation of Parkin to mitochondria or autophagic clearance of mitochondria. In addition, an SOD mimetic that attenuated the superoxide level suppressed mitophagy, while an SOD inhibitor accumulated cellular superoxide and promoted mitophagy. Furthermore, blockage of the p38 signaling pathway inhibited mitophagy induced by ROS, suggesting that it may contribute to the activation of ROS-mediated mitophagy. Together, our study sheds light on the link between ROS and mitophagy at a molecular level, and suggests the therapeutic potential of regulating mitophagy through the superoxide-p38-mitophagy axis.
Assuntos
Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Proteínas Quinases/metabolismo , Ionóforos de Próton/farmacologia , Superóxidos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antimicina A/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Mitofagia/efeitos dos fármacos , Doença de Parkinson/patologia , Transporte Proteico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.
Assuntos
Células da Granulosa/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Suínos , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Antimicina A/administração & dosagem , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Carbonil Cianeto m-Clorofenil Hidrazona/administração & dosagem , Carbonil Cianeto m-Clorofenil Hidrazona/análogos & derivados , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células Cultivadas , Feminino , Células da Granulosa/fisiologia , Oligomicinas/administração & dosagem , Oligomicinas/farmacologia , Oócitos/fisiologia , Ionóforos de Próton/administração & dosagem , Ionóforos de Próton/farmacologia , Espécies Reativas de Oxigênio , Desacopladores/administração & dosagem , Desacopladores/farmacologiaRESUMO
Apoptosis is considered a crucial part of the host defense system in oysters according to previous reports; however, the exact process by which this occurs remains unclear. Besides, mitochondrial apoptosis is the primary method of apoptosis in vertebrate cells, but has been poorly studied in invertebrates and is quite controversial. In this study, we investigated the molecular mechanism of mitochondrial apoptosis in the Pacific oyster Crassostrea gigas. Notably, we show that most key elements involved in the vertebrate mitochondrial apoptosis pathway - including mitochondrial outer membrane permeabilization, cytochrome c release, and caspase activation - are also present in C. gigas. In contrast, the lack of Bcl-2 homology 3-only subfamily members and apoptotic protease activating factor-1 (APAF-1) protein revealed evolutionary diversity from other phyla. Our results support that mitochondrial apoptosis in animals predates the emergence of vertebrates, but suggest that an unexpectedly diverse mitochondrial apoptosis pathway may exist in invertebrates. In addition, our work provided new clues for an improved understanding of how bivalve acclimate themselves to an inconstant environment.
Assuntos
Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Caspases/genética , Crassostrea/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Evolução Biológica , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Caspases/metabolismo , Sequência Conservada , Crassostrea/classificação , Crassostrea/efeitos dos fármacos , Crassostrea/efeitos da radiação , Citocromos c/metabolismo , Regulação da Expressão Gênica , Variação Genética , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Hemócitos/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/efeitos da radiação , Permeabilidade , Filogenia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ionóforos de Próton/farmacologia , Transdução de Sinais , Raios UltravioletaRESUMO
Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.
Assuntos
Trifosfato de Adenosina/metabolismo , Bioensaio/instrumentação , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio , Animais , Antimicina A/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Respiração Celular , Sobrevivência Celular , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Camundongos , Mitocôndrias/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Oligomicinas/farmacologia , Cultura Primária de Células , Ionóforos de Próton/farmacologia , Rotenona/farmacologia , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Desacopladores/farmacologiaRESUMO
A recent study showed that hypoxia activates a Ca2+-sensitive, Na+-permeable non-selective cation channel (NSC) in carotid body glomus cells. We studied the effects of mitochondrial inhibitors that increase Ca2+ influx via Ca2+ channel (Cav), and receptor agonists that release Ca2+ from endoplasmic reticulum (ER) on NSC. Mitochondrial inhibitors (NaCN, FCCP, H2S, NO) elevated [Ca2+]i and activated NSC. Angiotensin II and acetylcholine that elevate [Ca2+]i via the Gq-IP3 pathway activated NSC. However, endothelin-1 (Gq) and 5-HT (Gq) showed little or no effect on [Ca2+]i and did not activate NSC. Adenosine (Gs) caused a weak rise in [Ca2+]i but did not activate NSC. Dopamine (Gs) and γ-aminobytyric acid (Gi) were ineffective in raising [Ca2+]i and failed to activate NSC. Store-operated Ca2+ entry (SOCE) produced by depletion of Ca2+ stores with cyclopiazonic acid activated NSC. Our results show that Ca2+ entry via Cav, ER Ca2+ release and SOCE can activate NSC. Thus, NSC contributes to both voltage- and receptor-mediated excitation of glomus cells.
Assuntos
Cálcio/metabolismo , Corpo Carotídeo/citologia , Células Quimiorreceptoras/fisiologia , Canais Iônicos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Angiotensina II/farmacologia , Anilidas/farmacologia , Animais , Animais Recém-Nascidos , Cafeína/farmacologia , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Quimiorreceptoras/efeitos dos fármacos , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Hipóxia/fisiopatologia , Ionóforos de Próton/farmacologia , Ratos , Ratos Sprague-Dawley , S-Nitroso-N-Acetilpenicilamina/farmacologia , Cianeto de Sódio/farmacologia , Tiadiazóis/farmacologiaRESUMO
Haematopoietic stem cells (HSCs) differ from their committed progeny by relying primarily on anaerobic glycolysis rather than mitochondrial oxidative phosphorylation for energy production. However, whether this change in the metabolic program is the cause or the consequence of the unique function of HSCs remains unknown. Here we show that enforced modulation of energy metabolism impacts HSC self-renewal. Lowering the mitochondrial activity of HSCs by chemically uncoupling the electron transport chain drives self-renewal under culture conditions that normally induce rapid differentiation. We demonstrate that this metabolic specification of HSC fate occurs through the reversible decrease of mitochondrial mass by autophagy. Our data thus reveal a causal relationship between mitochondrial metabolism and fate choice of HSCs and also provide a valuable tool to expand HSCs outside of their native bone marrow niches.
Assuntos
Transporte de Elétrons/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/classificação , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Desacopladores/farmacologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Glicólise/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Ionóforos de Próton/farmacologia , Receptores de Superfície Celular/metabolismo , Nicho de Células-Tronco/genética , Irradiação Corporal TotalRESUMO
The ionophore monensin displays potent activities against several coccidian parasites of veterinary and medical importance including the opportunistic pathogen of humans, Toxoplasma gondii. While monensin is used widely in animals, toxicity impedes its use in humans. Nonetheless, given its potency, understanding its mode of action would reveal vulnerable aspects of the parasite that can be exploited for drug development. We previously established that monensin induces Toxoplasma to undergo cell cycle arrest and an autophagy-like cell death. Interestingly, these effects are dependent on the mitochondrion-localized TgMSH-1 protein, suggesting that monensin disrupts mitochondrial function. We demonstrate that monensin treatment results in decreased mitochondrial membrane potential and altered morphology. These effects are mitigated by the antioxidant compound N-acetyl-cysteine suggesting that monensin causes an oxidative stress, which was indeed the case based on direct detection of reactive oxygen species. Moreover, over-expression of the antioxidant proteins glutaredoxin and peroxiredoxin 2 protect Toxoplasma from the deleterious effects of monensin. Thus, our studies show that the effects of monensin on Toxoplasma are due to a disruption of mitochondrial function caused by the induction of an oxidative stress and implicate parasite redox biology as a viable target for the development of drugs against Toxoplasma and related pathogenic parasites.
Assuntos
Mitocôndrias/efeitos dos fármacos , Monensin/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Sequestradores de Radicais Livres/farmacologia , Expressão Gênica/efeitos dos fármacos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Complexo de Golgi/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Ionóforos de Próton/farmacologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxoplasma/genética , Toxoplasma/metabolismoRESUMO
Mitochondrial dynamics, including fission and fusion, control the morphology and function of mitochondria, and disruption of mitochondrial dynamics leads to Parkinson's disease, Alzheimer's disease, metabolic diseases, and cancers. Currently, many types of commercial mitochondria probes are available, but high excitation energy and low photo-stability render them unsuitable for tracking mitochondrial dynamics in living cells. Therefore, mitochondrial targeting agents that exhibit superior anti-photo-bleaching ability, deep tissue penetration and intrinsically high three-dimensional resolutions are urgently needed. Two-photon-excited compounds that use low-energy near-infrared excitation lasers have emerged as non-invasive tools for cell imaging. In this work, terpyridyl cyclometalated Ir(III) complexes (Ir1-Ir3) are demonstrated as one- and two-photon phosphorescent probes for real-time imaging and tracking of mitochondrial morphology changes in living cells.
Assuntos
Complexos de Coordenação/metabolismo , Substâncias Luminescentes/metabolismo , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Fótons , Esferoides Celulares/ultraestrutura , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Células HeLa , Humanos , Irídio/química , Substâncias Luminescentes/síntese química , Mitocôndrias/metabolismo , Imagem Molecular , Compostos Orgânicos/química , Ionóforos de Próton/farmacologia , Piridinas/química , Sirolimo/farmacologia , Esferoides Celulares/metabolismoRESUMO
One of the affected tissues in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), a tissue that consists of terminally differentiated cells and that accumulates damage over time. In all tissues, mitochondria (mt), which play an essential role in both cell health (energy) and death (initiator of apoptosis), undergo an aging process through the accumulation of mtDNA damage, changes in mitochondrial dynamics, a reduction in biogenesis, and mitophagy, leading to an overall reduction in mitochondrial energy production and other non-energy-related functions. Here we have compared energy metabolism in primary human RPE cells isolated from aborted fetus or aged donor eyes and grown as stable monolayers. H2O2 treatment resulted in the generation of reactive oxygen species and superoxide, an effect that was significantly augmented by age. Mitochondrial metabolism, as analyzed by Seahorse respirometry, revealed reduced mitochondrial oxygen consumption (ATP production) at baseline and a complete loss of reserve capacity in aged cells. Likewise, glycolysis was blunted in aged cells. Taken together, these studies showed that RPE cells derived from aged donor eyes are more susceptible to oxidative stress, and exhibit a loss in mitochondrial respiratory reserve capacity and a reduction in glycolysis. These data suggest that while old cells may have sufficient energy at rest, they cannot mount a stress response requiring additional ATP and reducing agents. In summary, these data support the hypothesis that mitochondria or energy metabolism is a valid target for therapy in AMD.
Assuntos
Envelhecimento/metabolismo , Metabolismo Energético , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Idoso , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxidantes/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Cultura Primária de Células , Ionóforos de Próton/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Superóxidos/metabolismoRESUMO
Mitochondrial dynamics change mitochondrial morphological features and numbers as a part of adaptive cellular metabolism, which is vital for most eukaryotic cells and organisms. A disease or even death of an animal can occur if these dynamics are disrupted. Using large-scale genetic screening in fruit flies, we previously found the gene mitoguardin (Miga), which encodes a mitochondrial outer-membrane protein and promotes mitochondrial fusion. Knockout mouse strains were generated for the mammalian Miga homologs Miga1 and Miga2. Miga1/2-/- females show greatly reduced quality of oocytes and early embryos and are subfertile. Mitochondria became clustered in the cytoplasm of oocytes from the germinal-vesicle stage to meiosis II; production of reactive oxygen species increased in mitochondria and caused damage to mitochondrial ultrastructures. Additionally, reduced ATP production, a decreased mitochondrial-DNA copy number, and lower mitochondrial membrane potential were detected in Miga1/2-/- oocytes during meiotic maturation. These changes resulted in low rates of polar-body extrusion during oocyte maturation, reduced developmental potential of the resulting early embryos, and consequently female subfertility. We provide direct evidence that MIGA1/2-regulated mitochondrial dynamics is crucial for mitochondrial functions, ensure oocyte maturation, and maintain the developmental potential.
Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Oócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Western Blotting , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células Cultivadas , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Ionóforos de Próton/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Impairment of the autophagy-lysosome pathway is implicated with the changes in α-synuclein and mitochondrial dysfunction observed in Parkinson's disease (PD). Damaged mitochondria accumulate PINK1, which then recruits parkin, resulting in ubiquitination of mitochondrial proteins. These can then be bound by the autophagic proteins p62/SQSTM1 and LC3, resulting in degradation of mitochondria by mitophagy. Mutations in PINK1 and parkin genes are a cause of familial PD. We found a significant increase in the expression of p62/SQSTM1 mRNA and protein following mitophagy induction in human neuroblastoma SH-SY5Y cells. p62 protein not only accumulated on mitochondria, but was also greatly increased in the cytosol. Increased p62/SQSMT1 expression was prevented in PINK1 knock-down cells, suggesting increased p62 expression was a consequence of mitophagy induction. The transcription factors Nrf2 and TFEB, which play roles in mitochondrial and lysosomal biogenesis, respectively, can regulate p62/SQSMT1. We report that both Nrf2 and TFEB translocate to the nucleus following mitophagy induction and that the increase in p62 mRNA levels was significantly impaired in cells with Nrf2 or TFEB knockdown. TFEB translocation also increased expression of itself and lysosomal proteins such as glucocerebrosidase and cathepsin D following mitophagy induction. We also report that cells with increased TFEB protein have significantly higher PGC-1α mRNA levels, a regulator of mitochondrial biogenesis, resulting in increased mitochondrial content. Our data suggests that TFEB is activated following mitophagy to maintain autophagy-lysosome pathway and mitochondrial biogenesis. Therefore, strategies to increase TFEB may improve both the clearance of α-synuclein and mitochondrial dysfunction in PD. Damaged mitochondria are degraded by the autophagy-lysosome pathway and is termed mitophagy. Following mitophagy induction, the transcription factors Nrf2 and TFEB translocate to the nucleus, inducing the transcription of genes encoding for autophagic proteins such as p62, as well as lysosomal and mitochondrial proteins. We propose that these events maintain autophagic flux, replenish lysosomes and replace mitochondria.
Assuntos
Lisossomos/metabolismo , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Neuroblastoma/patologia , Neuroblastoma/ultraestrutura , Ionóforos de Próton/farmacologia , RNA Interferente Pequeno/farmacologia , Receptores de Superfície Celular/metabolismo , Proteína Sequestossoma-1 , Fatores de Tempo , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn(2+) ] ([Zn(2+) ]i ) elevations, a low affinity (Kd = 39 µM at pH 6.1) ratiometric Zn(2+) probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn(2+) ]i elevations reaching 2 µM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn(2+) ]i increased further to 5.6 µM; analogous acid- and NEM-induced [Zn(2+) ]i elevations could also be detected but not calibrated, using the high affinity Zn(2+) probe FluoZin-3. The data indicate that NEM causes Zn(2+) release from ligands that chelate Zn(2+) at pH 6.1. ATP could also chelate Zn(2+) at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn(2+) ]i elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn(2+) ]i increases. These data suggest that the total amount of Zn(2+) accumulated in intracellular ATP-dependent stores (Zn(2+) -ATP complexes and organelles that accumulate Zn(2+) in an ATP-dependent manner) is negligible compared to the amount of Zn(2+) accumulated in the acid-sensitive intracellular ligands. In vitro, upon acidification, Zn(2+) -cysteine complexes release Zn(2+) and ATP chelates the released Zn(2+) . However, in vivo (cultured neurons), an ATP depletion failed to enhance acid-induced [Zn(2+) ]i elevations. These [Zn(2+) ]i elevations were calibrated using a low affinity ratiometric probe FuraZin-1; they reached 2 µM levels and increased to 5 µM when a thiol-reactive agent, N-ethylmaleimide, compromised Zn(2+) binding by cysteines.
Assuntos
Citosol/metabolismo , Neurônios/metabolismo , Zinco/metabolismo , Ácidos/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Quelantes/farmacologia , Cisteína , Citosol/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Etilenodiaminas , Etilmaleimida/farmacologia , Magnésio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Compostos Policíclicos , Ionóforos de Próton/farmacologiaRESUMO
The objectives of this study were to examine the effects of feedlot production systems with and without the use of a ß-adrenergic agonist compared to an all-natural production program on feedlot performance and carcass characteristics. Crossbred beef steers ( = 336; initial BW = 379 ± 8 kg) were randomized to 1 of 3 treatments in a randomized complete block design (RCBD; 14 steers/pen; 8 pens/treatment). Treatments consisted of an all-natural treatment (NAT), a conventional treatment (CONV), and a conventional treatment with a ß-agonist (CONV-Z). All treatments were fed the same basal diet with NAT cattle receiving no growth promoting technologies. The CONV and CONV-Z cattle were implanted with 40 mg of estradiol and 200 mg of trenbolone acetate (TBA) on d 0 and were fed 33 and 9 mg/kg of monensin and tylosin daily, respectively. The CONV-Z cattle were fed zilpaterol hydrochloride (ZH) at 6.76 mg/kg (90% DM basis) for the last 20 days on feed (DOF) There was no effect of treatment on DMI ( = 0.83); however, CONV-Z steers gained 3.8% faster (1.64 vs. 1.58 kg/d; < 0.01) and were 5.3% more efficient (0.160 vs. 0.152; < 0.01) than CONV steers, and CONV steers gained 32.8% faster (1.58 vs. 1.19 kg/d; < 0.01) and were 26.7% more efficient (0.152 vs. 0.120; < 0.01) than NAT steers. There was a 35.7% improvement in estimated carcass gain (1.29 vs. 0.95 kg/d; < 0.01) and a 32.6% improvement in carcass efficiency (0.126 vs. 0.095; < 0.01) for CONV-Z steers compared to NAT steers. Hot carcass weight was increased by 8 kg for CONV-Z steers compared to CONV steers (394 vs. 386 kg; = 0.05) and 46 kg compared to NAT steers (394 vs. 348 kg; < 0.01). Longissimus muscle area was increased by 3.6 cm for CONV-Z steers compared to CONV steers (92.29 vs. 88.67 cm; = 0.02) and 12.1 cm for CONV-Z steers compared to NAT steers (92.29 vs. 80.16 cm; < 0.01), resulting in a 9.6% unit increase in USDA yield grade (YG) 1 (15.14 vs. 5.52%; < 0.05) and a 21.6% unit reduction in USDA YG 3 for CONV-Z steers compared to CONV steers (30.70 vs. 52.32%; < 0.05). The CONV-Z steers had a lower marbling score compared to the other treatments (432; 0.01), resulting in an 11.7% unit increase (20.70 vs. 9.03%; < 0.05) in USDA Select carcasses compared to CONV steers. The results of this experiment show that CONV-Z and CONV production results in a significant improvement in feedlot performance and USDA YG compared to NAT.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Composição Corporal/efeitos dos fármacos , Bovinos/crescimento & desenvolvimento , Hormônios/farmacologia , Aumento de Peso/efeitos dos fármacos , Adrenérgicos/administração & dosagem , Adrenérgicos/farmacologia , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Composição Corporal/fisiologia , Bovinos/fisiologia , Dieta/veterinária , Estradiol/administração & dosagem , Estradiol/farmacologia , Hormônios/administração & dosagem , Masculino , Monensin/administração & dosagem , Monensin/farmacologia , Ionóforos de Próton/administração & dosagem , Ionóforos de Próton/farmacologia , Acetato de Trembolona/administração & dosagem , Acetato de Trembolona/farmacologia , Compostos de Trimetilsilil/administração & dosagem , Compostos de Trimetilsilil/farmacologia , Tilosina/administração & dosagem , Tilosina/farmacologia , Aumento de Peso/fisiologiaRESUMO
In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp.
Assuntos
Carcinoma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Corantes Fluorescentes/metabolismo , Neoplasias Ovarianas/metabolismo , Rodaminas/metabolismo , Neoplasias do Colo do Útero/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Absorção Fisiológica/efeitos dos fármacos , Animais , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Feminino , Radioisótopos de Flúor , Humanos , Imunossupressores/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Ionóforos de Próton/farmacologia , Traçadores Radioativos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rodamina 123/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczynski et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108-2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin.