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1.
Life Sci ; 254: 117812, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32428596

RESUMO

AIMS: Since the role of the major mitochondrial NAD+-dependent deacetylase, sirtuin 3 (Sirt3), is differential in cancer, opposite to the well-known tumor-suppressing effect of hyperoxia, this study aimed to investigate the role of Sirt3 in triple-negative breast cancer (TNBC) cell line MDA-MB-231 upon hyperoxic (95% O2) conditions. MAIN METHODS: MDA-MB-231 cells were stably transfected with Flag-tagged Sirt-3 or empty plasmid. Western blot and real-time PCR were used to monitor the expression of proteins or genes involved in mitochondrial biogenesis, metabolic regulation and antioxidant defense. Immunocytochemistry and confocal microscopy were used to confirm the cellular localization and abundance of proteins. Flow cytometry was used to analyze mitochondrial mass, potential and ROS production, and MTT test as a measure of metabolic activity. Mitotic index analysis, colony-forming unit assay, DNA damage and Annexin V-FITC analyses were used to assess the differences in the growth and apoptosis rate. KEY FINDINGS: Although Sirt3 seemed to improve mitochondrial properties by increasing mitochondrial mass and potential, metabolic activity (Warburg effect) and antioxidative defense (SOD2, Cat), it also increased mitochondrial ROS, induced DNA damage, timp-1 expression, formation of multinucleated cells and apoptosis, and finally markedly reduced the proliferation of MDA-MB-231 cells. All these effects were even more evident upon the hyperoxic treatment, thus pointing towards combined negative effect of Sirt3 and hyperoxia on MDA-MB-231 cells. SIGNIFICANCE: Both Sirt3 and hyperoxia, alone or in combination, have the potential to negatively affect the malignant properties of the MDA-MB-231 cells and should be further explored as a possible therapy for TNBC.


Assuntos
Sobrevivência Celular/fisiologia , Hiperóxia/fisiopatologia , Mitocôndrias/fisiologia , Sirtuína 3/fisiologia , Neoplasias de Mama Triplo Negativas/fisiopatologia , Anexinas/metabolismo , Apoptose/fisiologia , Carcinogênese , Linhagem Celular Tumoral , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Índice Mitótico , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/genética , Células-Tronco , Transfecção , Neoplasias de Mama Triplo Negativas/metabolismo
2.
Chemistry ; 26(14): 3173-3180, 2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-32083355

RESUMO

Development of novel bioimaging materials that exhibit organelle specific accumulation continues to be at the forefront of research interests and efforts. Among the various subcellular organelles, mitochondria, which are found in the cytoplasm of eukaryotic cells, are of particular interest in relation to their vital function. To date, most molecular probes that target mitochondria utilise delocalised lipophilic cations such as triphenylphosphonium and pyridinium. However, the use of such charged motifs is known to be detrimental to the working function of the mitochondrial transmembrane potential and there remains a strong case for development of neutral mitochondrial fluorescent probes. Herein, we demonstrate for the first time the exploitation of diketopyrrolopyrrole-based chemistries for the realisation of a neutral fluorescent probe that exhibits organelle specific accumulation within the mitochondria at the nanomolar level. The synthesised probe, which bears a neutral triphenylphosphine oxide moiety, exhibits a large Stokes shift and high fluorescence quantum yield in water, both highly sought-after properties in the development of bioimaging agents. In vitro studies reveal no interference with cell metabolism when tested for the human MCF7 breast cancer cell and nanomolar subcellular organelle colocalisation with commercially available mitochondrial staining agent Mitotracker Red. In light of its novelty, neutral structure and the preferential accumulation at nanomolar concentrations we anticipate this work to be of significant interest for the increasingly larger community devoted to the realisation of neutral mitochondrial selective systems and more widely to those engaged in the rational development of superior organic architectures in the biological field.


Assuntos
Corantes Fluorescentes/química , Cetonas/química , Mitocôndrias/metabolismo , Compostos Organofosforados/química , Pirróis/química , Técnicas Biossensoriais , Humanos , Luz , Células MCF-7 , Potencial da Membrana Mitocondrial/fisiologia , Estrutura Molecular , Imagem Óptica , Compostos Orgânicos/química , Relação Estrutura-Atividade
3.
DNA Cell Biol ; 39(4): 661-670, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32101022

RESUMO

Fibroblast growth factor 21 (FGF21) is a hormone-like member of the FGF family that is associated with cell death in atherosclerosis. However, its underlying mechanisms remain unclear. In this study, the effect of FGF21 on endothelial cell pyroptosis and its potential mechanisms were investigated. Results showed that FGF21 inhibits oxidized low-density lipoprotein (ox-LDL)-induced pyroptosis and related molecular expression in human umbilical vein endothelial cells (HUVECs). Mitochondrial function was damaged by ox-LDL and restored by FGF21. A mechanism proved that ubiquinol cytochrome c reductase core protein I (UQCRC1) was downregulated by ox-LDL and upregulated by FGF21. Further, the silencing of UQCRC1 aggravated HUVEC pyroptosis and impaired mitochondrial function and reactive oxygen species (ROS) production. Moreover, Tet methylcytosine dioxygenase (TET2) was involved in the regulation of UQCRC1 expression and pyroptosis. In summary, FGF21 inhibited ox-LDL-induced HUVEC pyroptosis through the TET2-UQCRC1-ROS pathway.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Lipoproteínas LDL/metabolismo , Piroptose/fisiologia , Aterosclerose/patologia , Sobrevivência Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/genética , Fatores de Crescimento de Fibroblastos/genética , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
4.
PLoS One ; 15(2): e0229332, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32092105

RESUMO

The placenta, a tissue that is metabolically active and rich in mitochondria, forms a critical interface between the mother and developing fetus. Oxidative stress within this tissue, derived from the dysregulation of reactive oxygen species (ROS), has been linked to a number of adverse fetal outcomes. While such outcomes have been associated with mitochondrial dysfunction, the causal role of mitochondrial dysfunction and mitochondrially generated ROS in altering the process of placentation remains unclear. In this study, mitochondrial complex I activity was attenuated using 10 nM rotenone to induce cellular oxidative stress by increasing mitochondrial ROS production in the BeWo choriocarcinoma cell line. Increased mitochondrial ROS resulted in a significant decrease in the transcripts which encode for proteins associated with fusion (GCM1, ERVW-1, and ERVFRD-1) resulting in a 5-fold decrease in the percentage of BeWo fusion. This outcome was associated with increased indicators of mitochondrial fragmentation, as determined by decreased expression of MFN2 and OPA1 along with an increase in a marker of mitochondrial fission (DRP1). Importantly, increased mitochondrial ROS also resulted in a 5.0-fold reduction of human placental lactogen (PL) and a 4.4-fold reduction of insulin like growth factor 2 (IGF2) transcripts; hormones which play an important role in regulating fetal growth. The pre-treatment of rotenone-exposed cells with 5 mM N-acetyl cysteine (NAC) resulted in the prevention of these ROS mediated changes in BeWo function and supports a central role for mitochondrial ROS signaling in the maintenance and function of the materno-fetal interface.


Assuntos
Mitocôndrias/metabolismo , Hormônios Placentários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trofoblastos/metabolismo , Fusão Celular , Células Cultivadas , Feminino , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Gravidez , Espécies Reativas de Oxigênio/farmacologia , Rotenona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trofoblastos/efeitos dos fármacos
5.
PLoS One ; 15(2): e0228848, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32050000

RESUMO

We investigated the relation of 99mTc-MIBI uptake to mitochondrial membrane potential (MMP) in cancer cell lines and patient-derived tumor cells (PDCs). In T47D and HT29 cells with low MDR1 expression, FCCP dose-dependently reduced MMP and 99mTc-MIBI accumulation in similar patterns with nearly perfect linear relationships. T47D and HT29 cells with high MDR1 expression had low 99mTc-MIBI accumulation that was minimally affected by FCCP dose. In these cells, verapamil markedly increased 99mTc-MIBI accumulation to magnitudes that were excessive compared to MMP increase. Decreased plasma membrane potential by verapamil and its recovery by FCCP suggested that enhanced 99mTc-MIBI transport through modified plasma membranes contributed to the excess accumulation. Evaluation of three different colon cancer PDCs with low to modest MDR1 expression verified that FCCP significantly suppressed MMP and similarly reduced 99mTc-MIBI accumulation. Verapamil partially recovered both MMP and 99mTc-MIBI accumulation that was lowered by FCCP. Importantly, a high linear correlation was found (r = 0.865) between 99mTc-MIBI accumulation and MMP in these cells. These findings indicate that low baseline 99mTc-MIBI uptake that is markedly increased by verapamil represents cancer cells with high levels of MDR1 expression. However, in cancer cells with low or modest levels of MDR1 expression that do not markedly increase 99mTc-MIBI uptake by verapamil, the magnitude of uptake is largely dependent on cellular MMP.


Assuntos
Transporte Biológico/fisiologia , Potencial da Membrana Mitocondrial/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tecnécio Tc 99m Sestamibi/metabolismo , Verapamil/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Células HT29 , Humanos , Compostos Radiofarmacêuticos/metabolismo , Células Tumorais Cultivadas
6.
Am J Physiol Cell Physiol ; 318(2): C439-C447, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31875695

RESUMO

Cardiovascular diseases remain the leading cause of death worldwide. Although major therapeutic progress has been made during the past decades, a better understanding of the underlying mechanisms will certainly help to improve patient's prognosis. In vitro models, particularly adult mouse cardiomyocytes, have been largely used; however, their fragility and large size are major obstacles to the use of flow cytometry. Conventional techniques, such as cell imaging, require the use of large numbers of animals and are time consuming. Here, we described a new, simple, and rapid one-day protocol using living adult mouse cardiomyocytes in suspension exposed to hypoxia-reoxygenation that allows a multilabeling analysis by flow cytometry. Several parameters can be measured by fluorescent probes labeling to assess cell viability (propidium iodide, calcein-AM, and Sytox Green), mitochondrial membrane potential [DilC1(5) and TMRM], reactive oxygen species production (MitoSOX Red), and mitochondrial mass (MitoTracker Deep Red). We address the robustness and sensitivity of our model using a cardioprotective agent, cyclosporine A. Overall, our new experimental set-up offers a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes exposed to hypoxia-reoxygenation. Our model might be interesting to investigate other cellular stresses (oxidative and inflammation) or to perform pharmacological screening.


Assuntos
Hipóxia Celular/fisiologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Oxigênio/metabolismo , Animais , Cardiotônicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citometria de Fluxo/métodos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo
7.
Pflugers Arch ; 471(11-12): 1551-1564, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31713764

RESUMO

Hydrogen sulfide (H2S) is endogenously produced in pancreatic ß cells and its level is elevated in diabetes. Here, we report that H2S affects insulin secretion via two mechanisms that converge on cytosolic free Ca2+ ([Ca2+]i), a key mediator of insulin exocytosis. Cellular calcium imaging, using Fura-2 or Fluo-4, showed that exposure of INS-1E cells to H2S (30-100 µM) reduced both [Ca2+]i levels (by 21.7 ± 2.3%) and oscillation frequency (p < 0.01, n = 4). Consistent with a role of plasma membrane KATP channels (plasma-KATP), the effects of H2S on [Ca2+]i were blocked by gliclazide (a blocker of plasma-KATP channels), but were mimicked by diazoxide (an activator of plasma-KATP channels). Surprisingly, when Ca2+ entry via plasma membrane was inhibited using Ca2+-free external solutions, H2S increased [Ca2+]i by 39.7 ± 3.6% suggesting Ca2+ release from intracellular stores. H2S-induced [Ca2+]i increases were abolished by either FCCP (which depletes Ca2+ stored in mitochondria) or cyclosporine A (an inhibitor of mitochondrial permeability transition pore, mPTP) suggesting that H2S induces Ca2+ release from mitochondria. Measurement of mitochondrial membrane potential (MMP) suggested that H2S causes MMP depolarization, which was blocked by cyclosporine A. Finally, insulin measurements by ELISA indicated that H2S decreased insulin release from INS-1E cells, but after plasma membrane Ca2+ entry was blocked by nifedipine, H2S-induced mitochondrial Ca2+ release is able to increase insulin release. Together, our results indicate that H2S has dual effects on insulin release suggesting that, with different metabolic conditions, H2S may differentially modulate the insulin release from pancreatic ß cells and play a role in ß cell dysfunction.


Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Homeostase/fisiologia , Sulfeto de Hidrogênio/metabolismo , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Canais KATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Exocitose/fisiologia , Insulina/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Ratos
8.
Int J Mol Sci ; 20(21)2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31717806

RESUMO

Huntington's disease (HD) is an inherited neurodegenerative disorder, caused by an abnormal polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Mitochondrial dysfunction and impairment of the ubiquitin-proteasome system (UPS) are hallmarks of HD neurons. The extraneural manifestations of HD are still unclear. We investigated the crosstalk between mitochondria and proteolytic function in skin fibroblasts from juvenile HD patients. We found reduced mitosis, increased cell size, elevated ROS and increased mitochondrial membrane potential in juvenile HD fibroblasts, while cellular viability was maintained. Mitochondrial OXPHOS analysis did not reveal significant differences compared to control. However, the level of mitochondrial fusion and fission proteins was significantly lower and branching in the mitochondria network was reduced. We hypothesized that juvenile HD fibroblasts counterbalance cellular damage and mitochondrial network deficit with altered proteasome activity to promote cell survival. Our data reveal that juvenile HD fibroblasts exhibit higher proteasome activity, which was associated with elevated gene and protein expression of parkin. Moreover, we demonstrate elevated proteasomal degradation of the mitochondrial fusion protein Mfn1 in diseased cells compared to control cells. Our data suggest that juvenile HD fibroblasts respond to mutant polyQ expansion of Htt with enhanced proteasome activity and faster turnover of specific UPS substrates to protect cells.


Assuntos
Fibroblastos/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , GTP Fosfo-Hidrolases/metabolismo , Glicólise , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Neurônios/metabolismo , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pele/citologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
9.
Med Sci Monit ; 25: 7547-7556, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31591376

RESUMO

BACKGROUND Cellular immunity plays a crucial role in sepsis, and lymphocyte apoptosis is a key factor in immune homeostasis. Tumor necrosis factor-alpha (TNF-alpha)-induced protein 8-like 2 (TIPE2) is suggested to play a critical role in maintaining immune homeostasis. This study investigated the role of TIPE2 in CD4⁺ T lymphocyte apoptosis based on a mouse model of thermal injury. MATERIAL AND METHODS BALB/c male mice were randomized into 6 groups: sham, burn, burn with siTIPE2, burn with siTIPE2 control, burn with TIPE2, and burn with TIPE2 control groups. Splenic CD4⁺ T lymphocytes were collected by use of a magnetic cell sorting system. RESULTS We found that TIPE2 downregulation reduced the CD4⁺ T lymphocytes apoptosis in the burn with siTIPE2 group, and the protein expression of P-smad2/P-Smad3 were remarkably downregulated. In the burn with siTIPE2 group, Bcl-2 expression was increased compared with that in the sham group (P<0.05), and Bim expression was reduced (P<0.05). In the burn with TIPE2 group, the mitochondrial membrane potential was markedly reduced (P<0.01), while cytochrome C expression was clearly higher than that in the other groups (P<0.01). Activities of caspase-3, -8, and -9 were notably higher in the burn with TIPE2 group relative to those for other groups (P<0.05). CONCLUSIONS Downregulation of TIPE2 in vivo can reduce the apoptosis of CD4⁺ T lymphocytes following thermal damage, and activate the TGFß downstream signaling of Smad2/Smad3, upregulating Bim, and downregulating Bcl-2.


Assuntos
Apoptose/efeitos dos fármacos , Queimaduras/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Queimaduras/fisiopatologia , Linfócitos T CD4-Positivos/metabolismo , Citocromos c/metabolismo , Temperatura Alta/efeitos adversos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , RNA Interferente Pequeno/genética , Sepse/imunologia , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
10.
Neurochem Res ; 44(11): 2577-2589, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541352

RESUMO

Cerebral ischemia is known to trigger a series of intracellular events such as changes in metabolism, membrane function and intracellular transduction, which eventually leads to cell death. Many of these processes are mediated by intracellular signaling cascades that involve protein kinase activation. Among all the kinases activated, the serine/threonine kinase family, protein kinase C (PKC), particularly, has been implicated in mediating cellular response to cerebral ischemic and reperfusion injury. In this study, using oxygen-glucose deprivation (OGD) in acute cortical slices as an in vitro model of cerebral ischemia, I show that PKC family of isozymes, specifically PKCγ and PKCε are differentially activated during OGD. Detecting the expression and activation levels of these isozymes in response to different durations of OGD insult revealed an early activation of PKCε and delayed activation of PKCγ, signifying their roles in response to different durations and stages of ischemic stress. Specific inhibition of PKCγ and PKCε significantly attenuated OGD induced cytotoxicity, rise in intracellular calcium, membrane depolarization and reactive oxygen species formation, thereby enhancing neuronal viability. This study clearly suggests that PKC family of isozymes; specifically PKCγ and PKCε are involved in OGD induced intracellular responses which lead to neuronal death. Thus isozyme specific modulation of PKC activity may serve as a promising therapeutic route for the treatment of acute cerebral ischemic injury.


Assuntos
Isquemia Encefálica/metabolismo , Córtex Cerebral/metabolismo , Proteína Quinase C-épsilon/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Animais , Cálcio/metabolismo , Hipóxia Celular/fisiologia , Glucose/deficiência , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo
11.
Cryobiology ; 90: 100-102, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31419408

RESUMO

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, -10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.


Assuntos
Crioprotetores/farmacologia , Epididimo/citologia , Preservação do Sêmen/métodos , Testículo/citologia , Vitrificação/efeitos dos fármacos , Reação Acrossômica/fisiologia , Adulto , Criopreservação/métodos , Transferência Embrionária , Feminino , Congelamento , Humanos , Nascimento Vivo , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Gravidez , Motilidade Espermática/fisiologia , Espermatozoides/fisiologia , Sacarose
12.
Nat Commun ; 10(1): 3732, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427612

RESUMO

Recently identified core proteins (MICU1, MCU, EMRE) forming the mitochondrial Ca2+ uniporter complex propelled investigations into its physiological workings. Here, we apply structured illumination microscopy to visualize and localize these proteins in living cells. Our data show that MICU1 localizes at the inner boundary membrane (IBM) due to electrostatic interaction of its polybasic domain. Moreover, this exclusive localization of MICU1 is important for the stability of cristae junctions (CJ), cytochrome c release and mitochondrial membrane potential. In contrast to MICU1, MCU and EMRE are homogeneously distributed at the inner mitochondrial membrane under resting conditions. However, upon Ca2+ elevation MCU and EMRE dynamically accumulate at the IBM in a MICU1-dependent manner. Eventually, our findings unveil an essential function of MICU1 in CJ stabilization and provide mechanistic insights of how sophistically MICU1 controls the MCU-Complex while maintaining the structural mitochondrial membrane framework.


Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Membranas Mitocondriais/metabolismo
13.
Cryobiology ; 90: 54-62, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31446003

RESUMO

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-ß-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm-oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.


Assuntos
Colesterol/farmacologia , Crioprotetores/farmacologia , Oócitos/crescimento & desenvolvimento , Oogênese/efeitos dos fármacos , Glicoproteínas da Zona Pelúcida/metabolismo , beta-Ciclodextrinas/farmacologia , Animais , Caspase 3/biossíntese , Caspase 8/biossíntese , Caspase 9/biossíntese , Criopreservação/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/fisiologia , Gravidez , Espermatozoides , Suínos , Ubiquitinação/efeitos dos fármacos , Vitrificação/efeitos dos fármacos , Zona Pelúcida/metabolismo
14.
Biomed Pharmacother ; 118: 108940, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31382130

RESUMO

Type 2 diabetes (T2D) appears to be a significant risk factor for brain injury. Glutaredoxin 2 (GRX2) belongs to the oxidoreductase family and plays an essential role in regulating various cellular processes. However, the pathogenic role of GRX2 in high fat diet (HFD)-induced brain injury is poorly understood. In the study, the loss-of-function approach was used to explore the effects of GRX2 on brain injury in HFD-challenged mice. The results indicated that HFD treatment resulted in significant increases in the change of body weight, insulin resistance and serum lipid deposition, which were markedly exaggerated by the loss of GRX2. Moreover, HFD-caused cognitive dysfunction was further promoted in GRX2 knockout mice. Histological analysis suggested that HFD administration led to the hippocampus damage, which was potentiated by GRX2 deficiency. In addition, GRX deletion enhanced HFD-induced inflammatory response in hippocampus of mice. Furthermore, GRX2 knockout markedly enhanced HFD-triggered insulin resistance in hippocampus of mice through down-regulating the protein levels of p-insulin receptor substrate 1 (IRS1) (Y632) and p-AKT (S473). The phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) suppressed by HFD administration was further reduced by GRX2 ablation. Moreover, HFD-induced oxidative stress and mitochondrial dysfunction were significantly aggravated in hippocampus of GRX2-knockout mice, which were largely dependent on the modulation of GSK-3ß signaling. These results above demonstrated that GRX2 was responsible for HFD-induced brain injury by enhancing insulin resistance, inflammation, oxidative stress and mitochondrial impairment via the meditation of GSK-3ß.


Assuntos
Lesões Encefálicas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glutarredoxinas/deficiência , Glicogênio Sintase Quinase 3 beta/metabolismo , Resistência à Insulina , Mitocôndrias/metabolismo , Animais , Astrócitos/metabolismo , Comportamento Animal/fisiologia , Lesões Encefálicas/etiologia , Células Cultivadas , Disfunção Cognitiva/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Teste de Tolerância a Glucose , Glicogênio Sintase Quinase 3 beta/genética , Inflamação , Potencial da Membrana Mitocondrial/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo
15.
Int J Mol Sci ; 20(13)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31323920

RESUMO

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca2+ levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression.


Assuntos
Sistemas CRISPR-Cas/fisiologia , Microglia/metabolismo , Receptores de GABA/metabolismo , Sistemas CRISPR-Cas/genética , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Fosforilação Oxidativa , Receptores de GABA/deficiência , Receptores de GABA/genética , Esteroides/metabolismo
16.
Int J Biol Sci ; 15(8): 1637-1653, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360107

RESUMO

Butein, a member of the chalcone family, is a potent anticarcinogen against multiple cancers, but its specific anti-NSCLC mechanism remains unknown. The present study examined the effects of butein treatment on NSCLC cell lines and NSCLC xenografts. Butein markedly decreased NSCLC cell viability; inhibited cell adhesion, migration, invasion, and colony forming ability; and induced cell apoptosis and G2/M phase arrest in NSCLC cells. Moreover, butein significantly inhibited PC-9 xenograft growth. Both in vivo and in vitro studies verified that butein exerted anti-NSCLC effect through activating endoplasmic reticulum (ER) stress-dependent reactive oxygen species (ROS) generation. These pro-apoptotic effects were reversed by the use of 4- phenylbutyric acid (4-PBA), CHOP siRNA, N-acetyl-L-cysteine (NAC) and Z-VAD-FMK (z-VAD) in vitro. Moreover, inhibition of ER stress markedly reduced ROS generation. In addition, in vivo studies further confirmed that inhibition of ER stress or oxidative stress partially abolished the butein-induced inhibition of tumor growth. Therefore, butein is a potential therapeutic agent for NSCLC, and its anticarcinogenic action might be mediated by ER stress-dependent ROS generation and the apoptosis pathway.


Assuntos
Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células A549 , Acetilcisteína/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Butilaminas/metabolismo , Adesão Celular/genética , Adesão Celular/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Camundongos Nus , Estresse Oxidativo/genética , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
17.
Methods Mol Biol ; 2029: 175-183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31273742

RESUMO

Ischemic heart disease is the leading cause of death worldwide. Stem cell therapy to repair and regenerate the infarcted myocardium is a promising approach to address this unmet medical need. However, the poor survival of transplanted cells in the hostile ischemic myocardium has been a major hurdle in achieving an effective cell therapy against myocardial infarction. As such, novel strategies to promote the survival of transplanted cells are highly sought after. Mitochondria are intimately involved in cell survival and have been the main organelles being targeted for cytoprotection. Mitochondrial morphology is linked to mitochondrial function and cell viability. Therefore, quantitative methodologies to obtain reliable and reproducible results of mitochondrial morphology and function are essential for identifying and developing new cytoprotective strategies to enhance the survival of stem cells post-transplantation. Here, we describe methods for assessing mitochondrial morphology, mitochondrial membrane potential, and mitochondrial reactive oxygen species production.


Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Células-Tronco/citologia , Sobrevivência Celular/fisiologia , Citoproteção/fisiologia , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Espécies Reativas de Oxigênio/metabolismo , Regeneração/fisiologia , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismo
18.
Zygote ; 27(4): 203-213, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31296276

RESUMO

The present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus-oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 µM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Células da Granulosa/fisiologia , Oócitos/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Dietilexilftalato/toxicidade , Feminino , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/citologia , Potencial da Membrana Mitocondrial/fisiologia , Oócitos/citologia , Oócitos/metabolismo , Estresse Oxidativo/fisiologia , Plastificantes/toxicidade , Ratos , Espécies Reativas de Oxigênio/metabolismo
19.
J Immunol ; 203(3): 639-646, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31209102

RESUMO

Signaling through CD27 plays a role in T cell activation and memory. However, it is currently unknown how this costimulatory receptor influences CD4+ effector T (Teff) cells in inflamed tissues. In the current study, we used a murine model of inducible self-antigen expression in the epidermis to elucidate the functional role of CD27 on autoreactive Teff cells. Expression of CD27 on Ag-specific Teff cells resulted in enhanced skin inflammation when compared with CD27-deficient Teff cells. CD27 signaling promoted the accumulation of IFN-γ and IL-2-producing T cells in skin draining lymph nodes in a cell-intrinsic fashion. Surprisingly, this costimulatory pathway had minimal effect on early T cell activation and proliferation. Instead, signaling through CD27 resulted in the progressive survival of Teff cells during the autoimmune response. Using BH3 profiling to assess mitochondrial cell priming, we found that CD27-deficient cells were equally as sensitive as CD27-sufficient cells to mitochondrial outer membrane polarization upon exposure to either BH3 activator or sensitizer peptides. In contrast, CD27-deficient Teff cells expressed higher levels of active caspase 8. Taken together, these results suggest that CD27 does not promote Teff cell survival by increasing expression of antiapoptotic BCL2 family members but instead acts by preferentially suppressing the cell-extrinsic apoptosis pathway, highlighting a previously unidentified role for CD27 in augmenting autoreactive Teff cell responses.


Assuntos
Autoantígenos/imunologia , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Epiderme/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Transferência Adotiva , Animais , Apoptose/fisiologia , Autoimunidade/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Memória Imunológica/imunologia , Inflamação/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Animais , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética
20.
Mediators Inflamm ; 2019: 1897820, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182930

RESUMO

In our previous study, lipopolysaccharide (LPS) significantly reduced the cell viability of primary bovine mammary epithelial cells (bMEC) leading to cell apoptosis, which were prevented by caffeic acid (CA) through inhibiting NF-κB activation and reducing proinflammatory cytokine expression. While the underlying mechanism remains unclear, here, we determined that LPS induced the extensive microstructural damage of bMEC, especially the mitochondria and endoplasmic reticulum. Then, the obvious reduction of mitochondrial membrane potential and expression changes of apoptosis-associated proteins (Bcl-2, Bax, and casepase-3) indicated that apoptosis signaling through the mitochondria should be responsible for the cell viability decrease. Next, the high-throughput cDNA sequencing (RNA-Seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were employed to verify that the MAPK and JAK-STAT signaling pathways also were the principal targets of LPS. Following, the critical proteins (ERK, JNK, p38, and c-jun) of the MAPK signaling pathways were activated, and the release of proinflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) regulated by NF-κB and MAPKs was significantly increased, which can promote a cascade of inflammation that induces cell injury and apoptosis. Meanwhile, CA significantly inhibited the activation of MAPKs and the release of proinflammatory cytokines in a dose-dependent manner, which were similar to its effects on the NF-κB activation that we previously published. So we concluded that CA regulates the proteins located in the upstream of multiple cell signal pathways which can reduce the LPS-induced activation of NF-κB and MAPKs, thus weakening the inflammatory response and maintaining cell structure and function, which accordingly inhibit apoptosis.


Assuntos
Ácidos Cafeicos/farmacologia , Células Epiteliais/metabolismo , Lipopolissacarídeos/farmacologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Bovinos , DNA Complementar/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
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