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1.
J Biol Chem ; 298(9): 102336, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35931111

RESUMO

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.


Assuntos
Corantes Fluorescentes , Quelantes de Ferro , Mitocôndrias , Animais , Cálcio/metabolismo , Cátions Bivalentes/análise , Células Cultivadas , Fluorescência , Corantes Fluorescentes/química , Quelantes de Ferro/análise , Camundongos , Mitocôndrias/química , Proteínas Mitocondriais/química , Oxiquinolina/química , Rodamina 123 , Tapsigargina/farmacologia
2.
Cancer Res ; 82(10): 1969-1990, 2022 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-35404405

RESUMO

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.


Assuntos
Monóxido de Carbono , eIF-2 Quinase , Apoptose , Autofagia , Monóxido de Carbono/farmacologia , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Linfócitos T/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
3.
Cancers (Basel) ; 13(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34062962

RESUMO

Polyploid giant cancer cells (PGCC) constitute a transiently senescent subpopulation of cancer cells that arises in response to stress. PGCC are capable of generating progeny via a primitive, cleavage-like cell division that is dependent on the sphingolipid enzyme acid ceramidase (ASAH1). The goal of this study was to understand differences in sphingolipid metabolism between non-polyploid and polyploid cancer cells to gain an understanding of the ASAH1-dependence in the PGCC population. Steady-state and flux analysis of sphingolipids did not support our initial hypothesis that the ASAH1 product sphingosine is rapidly converted into the pro-survival lipid sphingosine-1-phosphate. Instead, our results suggest that ASAH1 activity is important for preventing the accumulation of long chain ceramides such as C16-ceramide. We therefore determined how modulation of C16-ceramide, either through CerS6 or p53, a known PGCC suppressor and enhancer of CerS6-derived C16-ceramide, affected PGCC progeny formation. Co-expression of the CerS6 and p53 abrogated the ability of PGCC to form offspring, suggesting that the two genes form a positive feedback loop. CerS6 enhanced the effect of p53 by significantly increasing protein half-life. Our results support the idea that sphingolipid metabolism is of functional importance in PGCC and that targeting this signaling pathway has potential for clinical intervention.

4.
Hepatol Commun ; 5(6): 976-991, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34141984

RESUMO

The electron transfer flavoprotein (ETF) complex, made up of the ETF alpha subunit (ETFA), ETF beta subunit (ETFB), and ETF dehydrogenase (ETFDH), regulates fatty acid ß-oxidation activity while scavenging leaked electrons through flavin adenine dinucleotide (FAD)/reduced form FAD (FADH2) redox reactions in mitochondria. Here, we hypothesized that ETF dysfunction-mediated FAD deficiency may result in increased mitochondrial oxidative stress and steatosis and subsequent liver injury. We report that etfa haploinsufficiency caused hyperlipidemia, hypercholesterolemia, and hepatic steatosis and injury in adult zebrafish. Further, etfa+/ - mutant livers had reduced levels of FAD and glutathione and an increase in reactive oxygen species. Because FAD depletion might be critical in the pathogenesis of the liver lesion identified in etfa+/ - mutants, we used riboflavin to elevate FAD levels in the liver and found that riboflavin supplementation significantly suppressed hepatic steatosis and injury in etfa+/ - mutants through suppression of oxidative stress and de novo lipogenesis in the liver. Additionally, we found that adenosine triphosphate-linked mitochondrial oxygen consumption and mitochondrial membrane potential were reduced in etfa+/ - primary hepatocytes and that riboflavin supplementation corrected these defects. Conclusion: FAD depletion caused by etfa haploinsufficiency plays a key role in hepatic steatosis and oxidative stress-mediated hepatic injury in adult zebrafish. This raises the possibility that people with ETFA haploinsufficiency have a high risk for developing liver disease.

5.
Methods Mol Biol ; 2277: 423-431, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080166

RESUMO

Intracellular Ca2+ is strictly regulated to maintain optimal levels for function of cellular organelles as well as mitochondrial respiratory signaling at the tricarboxylic acid cycle and electron transport chain level. Optimal Ca2+ concentration for these processes vary between cell types. Furthermore, exposure of mitochondria to sustained, elevated levels of Ca2+ induces mitochondrial Ca2+ overload and damage to mitochondrial oxidative phosphorylation and ATP production. Isolated mitochondria are widely used to study mitochondrial physiology and drug effects on mitochondrial metabolism and respiratory function. However, isolated mitochondria are easily damaged during the mitochondrial isolation process. The present article describes a mitochondrial isolation method using Ca2+-chelation to minimize mitochondrial damage. We follow up the isolation process with an application that requires an optimized buffer Ca2+ concentration: the characterization of their respiratory function using a high-resolution respirometric assay.


Assuntos
Técnicas Citológicas/métodos , Mitocôndrias/metabolismo , Biologia Molecular/métodos , Epitélio Pigmentado da Retina/citologia , Trifosfato de Adenosina/metabolismo , Quelantes de Cálcio/farmacologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Biologia Molecular/instrumentação , Epitélio Pigmentado da Retina/efeitos dos fármacos
6.
Cell Rep ; 35(5): 109076, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33951438

RESUMO

We lack a mechanistic understanding of aging-mediated changes in mitochondrial bioenergetics and lipid metabolism that affect T cell function. The bioactive sphingolipid ceramide, induced by aging stress, mediates mitophagy and cell death; however, the aging-related roles of ceramide metabolism in regulating T cell function remain unknown. Here, we show that activated T cells isolated from aging mice have elevated C14/C16 ceramide accumulation in mitochondria, generated by ceramide synthase 6, leading to mitophagy/mitochondrial dysfunction. Mechanistically, aging-dependent mitochondrial ceramide inhibits protein kinase A, leading to mitophagy in activated T cells. This aging/ceramide-dependent mitophagy attenuates the antitumor functions of T cells in vitro and in vivo. Also, inhibition of ceramide metabolism or PKA activation by genetic and pharmacologic means prevents mitophagy and restores the central memory phenotype in aging T cells. Thus, these studies help explain the mechanisms behind aging-related dysregulation of T cells' antitumor activity, which can be restored by inhibiting ceramide-dependent mitophagy.


Assuntos
Envelhecimento , Ceramidas/metabolismo , Mitocôndrias/metabolismo , Linfócitos T/metabolismo , Animais , Humanos , Camundongos , Transdução de Sinais
7.
Front Immunol ; 12: 628062, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746964

RESUMO

Complement component 3 fragment C3a is an anaphylatoxin involved in promoting cellular responses important in immune response and host defense. Its receptor (C3a receptor, C3aR) is distributed on the plasma membrane; however, lysosomal localization in immune cells has been reported. Oxidative stress increases intracellular reactive oxygen species (ROS), and ROS activate complement signaling in immune cells and metabolic reprogramming. Here we tested oxidative stress and intracellular complement in mitochondrial dysfunction in RPE cells using high resolution live-cell imaging, and metabolism analysis in isolated mitochondria using Seahorse technology. While C3aR levels were unaffected by oxidative stress, its cell membrane levels decreased and mitochondrial (mt) localization increased. Trafficking was dependent on endocytosis, utilizing endosomal-to-mitochondrial cargo transfer. H2O2-treatment also increased C3a-mtC3aR co-localization dose-dependently. In isolated mitochondria from H2O2-treated cells C3a increased mitochondrial Ca2+ uptake, that could be inhibited by C3aR antagonism (SB290157), mitochondrial Ca2+ uniporter blocker (Ru360), and Gαi-protein inhibition (pertussis toxin, PTX); and inhibited mitochondrial repiration in an SB290157- and PTX-dependent manner. Specifically, mtC3aR activation inhibited state III ADP-driven respiration and maximal respiratory capacity. Mitochondria from control cells did not respond to C3a. Furthermore, transmitochondrial cybrid ARPE-19 cells harboring J haplogroup mitochondria that confer risk for age-related macular degeneration, showed high levels of mtC3aR and reduced ATP production upon C3a stimulation. Our findings suggest that oxidative stress increases mtC3aR, leading to altered mitochondrial calcium uptake and ATP production. These studies will have important implication in our understanding on the balance of extra- and intracellular complement signaling in controlling cellular health and dysfunction.


Assuntos
Metabolismo Energético , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Receptores de Complemento/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Linhagem Celular , Respiração Celular , Endocitose , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Peróxido de Hidrogênio/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Estresse Oxidativo/efeitos dos fármacos , Transporte Proteico , Receptores de Complemento/genética , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/imunologia
8.
Biol Cell ; 113(3): 133-145, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33275284

RESUMO

BACKGROUND INFORMATION: Wnt/ß-catenin signalling, in the microenvironment of pluripotent stem cells (PSCs), plays a critical role in their differentiation and proliferation. Contradictory reports on the role of Wnt/ß-catenin signalling in PSCs self-renewal and differentiation, however, render these mechanisms largely unclear. RESULTS: Wnt/ß-catenin signalling pathway in human-induced pluripotent stem cells (hiPSCs) was activated by inhibiting glycogen synthase kinase 3 (GSK3), driving the cells into a mesodermal/mesenchymal state, exhibiting proliferative, invasive and anchorage-independent growth properties, where over 70% of cell population became CD 44 (+)/CD133 (+). Wnt/ß-catenin signalling activation also altered the metabolic state of hiPSCs from aerobic glycolysis to oxidative metabolism and changed their drug and oxidative stress sensitivities. These effects of GSK3 inhibition were suppressed in HIF1α-stabilised cells. CONCLUSIONS: Persistent activation of Wnt/ß-catenin signalling endows hiPSCs with proliferative/invasive 'teratoma-like' states, shifting their metabolic dependence and allowing HIF1α-stabilisation to inhibit their proliferative/invasive properties. SIGNIFICANCE: The hiPSC potential to differentiate into 'teratoma-like' cells suggest that stem cells may exist in two states with differential metabolic and drug dependency.


Assuntos
Células-Tronco Pluripotentes Induzidas , Via de Sinalização Wnt/fisiologia , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo
9.
J Biol Chem ; 295(52): 18091-18104, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33087445

RESUMO

Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential, and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis, showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that, sildenafil, a phosphodiesterase 5 inhibitor, induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content, and voltage-dependent anion channel protein expression. Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil depends upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/química , Endotélio Vascular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Núcleo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Mitocôndrias/patologia , Biogênese de Organelas , Consumo de Oxigênio , Fatores de Transcrição/genética
10.
Front Pharmacol ; 11: 577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32457608

RESUMO

Gynecologic cancers are among the most lethal cancers found in women, and, advanced stage cancers are still a treatment challenge. Ion channels are known to contribute to cellular homeostasis in all cells and mounting evidence indicates that ion channels could be considered potential therapeutic targets against cancer. Nevertheless, the pharmacologic effect of targeting ion channels in cancer is still understudied. We found that the expression of Kir6.2/SUR2 potassium channel is a potential favorable prognostic factor in gynecologic cancers. Also, pharmacological stimulation of the Kir6.2/SUR2 channel activity with the selective activator molecule minoxidil arrests tumor growth in a xenograft model of ovarian cancer. Investigation on the mechanism linking the Kir6.2/SUR2 to tumor growth revealed that minoxidil alters the metabolic and oxidative state of cancer cells by producing mitochondrial disruption and extensive DNA damage. Consequently, application of minoxidil results in activation of a caspase-3 independent cell death pathway. Our data show that repurposing of FDA approved K+ channel activators may represent a novel, safe adjuvant therapeutic approach to traditional chemotherapy for the treatment of gynecologic cancers.

11.
Cell Rep ; 28(7): 1879-1893.e7, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31412253

RESUMO

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.


Assuntos
Reprogramação Celular , Regulação Neoplásica da Expressão Gênica , Lisofosfolipídeos/metabolismo , Melanoma Experimental/patologia , PPAR gama/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Esfingosina/análogos & derivados , Linfócitos T/imunologia , Animais , Feminino , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação Oxidativa , Receptores de Lisoesfingolipídeo/metabolismo , Transdução de Sinais , Esfingosina/metabolismo , Linfócitos T/metabolismo
12.
FASEB J ; 33(7): 8186-8201, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30951369

RESUMO

Statins, widely used to treat hypercholesterolemia, inhibit the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme of de novo cholesterol (Chol) synthesis. Statins have been also reported to slow tumor progression. In cancer cells, ATP is generated both by glycolysis and oxidative phosphorylation. Mitochondrial membrane potential (ΔΨ), a readout of mitochondrial metabolism, is sustained by the oxidation of respiratory substrates in the Krebs cycle to generate NADH and flavin adenine dinucleotide, which are further oxidized by the respiratory chain. Here, we studied the short-term effects of statins (3-24 h) on mitochondrial metabolism on cancer cells. Lovastatin (LOV) and simvastatin (SIM) increased ΔΨ in HepG2 and Huh7 human hepatocarcinoma cells and HCC4006 human lung adenocarcinoma cells. Mitochondrial hyperpolarization after LOV and SIM was dose and time dependent. Maximal increase in ΔΨ occurred at 10 µM and 24 h for both statins. The structurally unrelated atorvastatin also hyperpolarized mitochondria in HepG2 cells. Cellular and mitochondrial Chol remained unchanged after SIM. Both LOV and SIM decreased basal respiration, ATP-linked respiration, and ATP production. LOV and SIM did not change the rate of lactic acid production. In summary, statins modulate mitochondrial metabolism in cancer cells independently of the Chol content in cellular membranes without affecting glycolysis.-Christie, C. F., Fang, D., Hunt, E. G., Morris, M. E., Rovini, A., Heslop, K. A., Beeson, G. C., Beeson, C. C., Maldonado, E. N. Statin-dependent modulation of mitochondrial metabolism in cancer cells is independent of cholesterol content.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Carcinoma Hepatocelular/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Hepáticas/metabolismo , Lovastatina/farmacologia , Neoplasias Pulmonares/metabolismo , Mitocôndrias Hepáticas/metabolismo , Sinvastatina/farmacologia , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/patologia
13.
Anal Biochem ; 577: 52-58, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31022379

RESUMO

Optimum cytosolic calcium concentrations support balanced mitochondrial respiration. However, cytosolic Ca2+ concentrations vary among cell types and excess Ca2+ can cause mitochondrial dysfunction. We optimized an isolation protocol to eliminate excess Ca2+ and thereby minimizing structural damage. Ca2+ uptake was monitored by measuring mitochondrial Ca2+-dependent PKA activity using cAMP ELISAs, and O2 consumption levels during mitochondrial respiration using high-resolution respirometry. 3 nM Ca2+ was found to increase cAMP levels and produce optimal state III respiration. Hence, optimized isolation of mitochondria from cell lines using calcium denudation provides the best platform for the study of Ca2+-dependent regulation of mitochondrial signaling.


Assuntos
Cálcio , Mitocôndrias/metabolismo , Cálcio/análise , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , AMP Cíclico/metabolismo , Citosol/química , Humanos , Oxigênio/metabolismo
14.
Nat Commun ; 10(1): 1296, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30899002

RESUMO

The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Glutamina/metabolismo , Hipoglicemiantes/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzenoacetamidas/farmacologia , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/antagonistas & inibidores , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Camundongos , Terapia de Alvo Molecular , Fenformin/farmacologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Tiadiazóis/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cell Rep ; 25(6): 1469-1484.e5, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30404003

RESUMO

Patients with mtDNA depletion syndrome 3 (MTDPS3) often die as children from liver failure caused by severe reduction in mtDNA content. The identification of treatments has been impeded by an inability to culture and manipulate MTDPS3 primary hepatocytes. Here we generated DGUOK-deficient hepatocyte-like cells using induced pluripotent stem cells (iPSCs) and used them to identify drugs that could improve mitochondrial ATP production and mitochondrial function. Nicotinamide adenine dinucleotide (NAD) was found to improve mitochondrial function in DGUOK-deficient hepatocyte-like cells by activating the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α). NAD treatment also improved ATP production in MTDPS3-null rats and in hepatocyte-like cells that were deficient in ribonucleoside-diphosphate reductase subunit M2B (RRM2B), suggesting that it could be broadly effective. Our studies reveal that DGUOK-deficient iPSC-derived hepatocytes recapitulate the pathophysiology of MTDPS3 in culture and can be used to identify therapeutics for mtDNA depletion syndromes.


Assuntos
DNA Mitocondrial/genética , Hepatócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , NAD/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Respiração Celular , Feminino , Glucose/metabolismo , Glicólise , Hepatócitos/citologia , Hepatócitos/ultraestrutura , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mutação/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ratos , Ribonucleotídeo Redutases/metabolismo , Síndrome
16.
Cell Metab ; 27(1): 85-100.e8, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29129787

RESUMO

Heightened effector function and prolonged persistence, the key attributes of Th1 and Th17 cells, respectively, are key features of potent anti-tumor T cells. Here, we established ex vivo culture conditions to generate hybrid Th1/17 cells, which persisted long-term in vivo while maintaining their effector function. Using transcriptomics and metabolic profiling approaches, we showed that the enhanced anti-tumor property of Th1/17 cells was dependent on the increased NAD+-dependent activity of the histone deacetylase Sirt1. Pharmacological or genetic inhibition of Sirt1 activity impaired the anti-tumor potential of Th1/17 cells. Importantly, T cells with reduced surface expression of the NADase CD38 exhibited intrinsically higher NAD+, enhanced oxidative phosphorylation, higher glutaminolysis, and altered mitochondrial dynamics that vastly improved tumor control. Lastly, blocking CD38 expression improved tumor control even when using Th0 anti-tumor T cells. Thus, strategies targeting the CD38-NAD+ axis could increase the efficacy of anti-tumor adoptive T cell therapy.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Imunoterapia , NAD/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Proteína Forkhead Box O1/metabolismo , Glutamina/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Sirtuína 1/metabolismo , Células Th1/imunologia , Células Th17/imunologia
17.
Am J Cardiovasc Dis ; 6(2): 46-54, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27335690

RESUMO

UNLABELLED: Estrogen-related Receptors (ERR) are members of the steroid hormone receptor superfamily of transcription factors that regulate expression of genes required for energy metabolism including mitochondrial biogenesis, fatty acid oxidation and oxidative phosphorylation. While ERRα and EPPγ isoforms are known to share a wide array of target genes in the adult myocardium, the function of ERRß has not been characterized in cardiomyocytes. The purpose of this study was to determine the role of ERRß in regulating energy metabolism in adult cardiomyocytes in primary culture. Adult feline cardiomyocytes were electrically stimulated to contract in either hypoxia (0.5% O2) or normoxia (21% O2). As compared to baseline values measured in normoxia, ERRß mRNA levels increased significantly after 8 hours of hypoxia and remained elevated over 24 h. Conversely, ERRß mRNA decreased to normoxic levels after 4 hours of reoxygenation. Hypoxia increased expression of the α and ß isoforms of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) mRNA by 6-fold and 3-fold, respectively. Knockdown of ERRß expression via adenoviral-mediated delivery of ERRß shRNA blocked hypoxia-induced increases in PGC-1ß mRNA, but not PGC-1α mRNA. Loss of ERRß had no effect on mtDNA content as measured after 24 h of hypoxia. To determine whether loss of ERRß affected mitochondrial function, oxygen consumption rates (OCR) were measured in contracting versus quiescent cardiomyocytes in normoxia. OCR was significantly lower in contracting cardiomyocytes expressing ERRß shRNA than scrambled shRNA controls. Maximal OCR also was reduced by ERRß knockdown. IN CONCLUSION: 1) hypoxia increases in ERRß mRNA expression in contracting cardiomyocytes; 2) ERRß is required for induction of the PGC-1ß isoform in response to hypoxia; 3) ERRß expression is required to sustain OCR in normoxic conditions.

18.
Int J Cardiol ; 187: 393-400, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25841134

RESUMO

BACKGROUND AND OBJECTIVES: In adult myocardium, Estrogen-Related Receptor α (ERRα) programs energetic capacity of cardiomyocytes by regulating expression of target genes required for mitochondrial biogenesis, fatty acid metabolism and oxidative phosphorylation. Transcriptional activation by ERRα is dependent on the α or ß isoform of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1). This study utilized a model of continuously contracting adult cardiomyocytes to determine the effects of sustained oxygen reduction (hypoxia) on ERRα target gene expression. METHODS AND RESULTS: Adult feline cardiomyocytes in primary culture were electrically stimulated to contract at 1 Hz in either normoxia (21% O2) or hypoxia (0.5% O2). Compared to normoxia, hypoxia increased PGC-1α mRNA and PGC-1ß mRNA levels by 16-fold and 14-fold after 24h. ERRα mRNA levels were increased 3-fold by hypoxia over the same time period. Treatment of cardiomyocytes with XCT-790, an ERRα inverse agonist, caused knockdown of ERRα protein expression. The increases in PGC-1 mRNA levels in response to hypoxia were blocked by XCT-790 treatment, which indicates that expression of PGC-1 isoforms is dependent on ERRα activity. The products of two ERRα target genes required for energy metabolism, Cox6c mRNA and Fabp3 mRNA, increased by 4.5-fold and 3.5 fold after 24h of hypoxia as compared to normoxic controls. These increases were blocked by XCT-790 treatment of hypoxic cardiomyocytes with a concomitant decrease in ERRα expression. CONCLUSIONS: ERRα activity is required to increase expression of PGC-1 isoforms and downstream target genes as part of the adaptive response of contracting adult cardiomyocytes to sustained hypoxia.


Assuntos
Miócitos Cardíacos/metabolismo , Receptores de Estrogênio/fisiologia , Fatores de Transcrição/biossíntese , Fatores Etários , Animais , Gatos , Estimulação Elétrica , Hipóxia , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Isoformas de Proteínas , Receptor ERRalfa Relacionado ao Estrogênio
19.
Cancer Res ; 74(21): 6036-6047, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25164014

RESUMO

Ex vivo-expanded CD8(+) T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced antitumor efficacy, relative to CD8(+) T cells with a central memory-like phenotype (TCM cells). Furthermore, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM cells possess relatively greater antioxidative capacity than TEM cells. Here, we report that TCM cells exhibit a relative increase compared with TEM cells in the expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity, and glycolytic enzyme levels. Notably, T-cell receptor-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, upregulated thiol levels and antioxidant genes. A comparison of antitumor CD8(+) T-cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth and that profiling this biomarker may have benefits to adoptive T-cell immunotherapy protocols.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Imunoterapia Adotiva , Neoplasias/imunologia , Compostos de Sulfidrila/metabolismo , Linfócitos T CD8-Positivos/imunologia , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Memória Imunológica/imunologia , Ativação Linfocitária , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia
20.
Chem Res Toxicol ; 26(9): 1323-32, 2013 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-23895456

RESUMO

A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for 1 or 5 h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader, and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled the identification of 20 compounds as uncouplers. This comprehensive approach allows for the evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs.


Assuntos
Poluentes Ambientais/toxicidade , Ensaios de Triagem em Larga Escala , Membranas Mitocondriais/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas
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