RESUMO
Acetylation of proteins seems a widespread process found in the three domains of life. Several studies have shown that besides histones, acetylation of lysine residues also occurs in non-nuclear proteins. Hence, it has been suggested that this covalent modification is a mechanism that might regulate diverse metabolic pathways by modulating enzyme activity, stability, and/or subcellular localization or interaction with other proteins. However, protein acetylation levels seem to have low correlation with modification of enzyme activity and pathway fluxes. In addition, the results obtained with mutant enzymes that presumably mimic acetylation have frequently been over-interpreted. Moreover, there is a generalized lack of rigorous enzyme kinetic analysis in parallel to acetylation level determinations. The purpose of this review is to analyze the current findings on the impact of acetylation on metabolic enzymes and its repercussion on metabolic pathways function/regulation.
Assuntos
Redes e Vias Metabólicas , Processamento de Proteína Pós-Traducional , Acetilação , Histonas , CinéticaRESUMO
The resveratrol (RSV) efficacy to affect the proliferation of several cancer cell lines was initially examined. RSV showed higher potency to decrease growth of metastatic HeLa and MDA-MB-231 (IC50â¯=â¯200-250⯵M) cells than of low metastatic MCF-7, SiHa and A549 (IC50â¯=â¯400-500⯵M) and non-cancer HUVEC and 3T3 (IC50≥600⯵M) cells after 48â¯h exposure. In order to elucidate the biochemical mechanisms underlying RSV anti-cancer effects, the energy metabolic pathways and the oxidative stress metabolism were analyzed in HeLa cells as metastatic-type cell model. RSV (200⯵M/48â¯h) significantly decreased both glycolysis and oxidative phosphorylation (OxPhos) protein contents (30-90%) and fluxes (40-70%) vs. non-treated cells. RSV (100⯵M/1-5â¯min) also decreased at a greater extent OxPhos flux (net ADP-stimulated respiration) of isolated tumor mitochondria (> 50%) than of non-tumor mitochondria (< 50%), particularly with succinate as oxidizable substrate. In addition, RSV promoted an excessive cellular ROS (2-3 times) production corresponding with a significant decrement in the SOD activity (but not in its content) and GSH levels; whereas the catalase, glutahione reductase, glutathione peroxidase and glutathione-S-transferase activities (but not their contents) remained unchanged. RSV (200⯵M/48â¯h) also induced cellular death although not by apoptosis but rather by promoting a strong mitophagy activation (65%). In conclusion, RSV impaired OxPhos by inducing mitophagy and ROS over-production, which in turn halted metastatic HeLa cancer cell growth.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Resveratrol/farmacologia , Células 3T3 , Animais , Linhagem Celular Tumoral , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos , Mitofagia/efeitos dos fármacos , Metástase Neoplásica/prevenção & controle , Compostos Fitoquímicos/farmacologiaRESUMO
To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes.
Assuntos
Proteínas Arqueais/metabolismo , Frutose-Bifosfatase/metabolismo , Methanosarcina/metabolismo , Fosfofrutoquinase-1/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Galinhas , Frutose-Bifosfatase/genética , Frutose-Bifosfatase/isolamento & purificação , Frutosefosfatos/metabolismo , Genes Arqueais , Cinética , Methanosarcina/genética , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/isolamento & purificação , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Cancer stem cells are responsible for tumor recurrence and metastasis. A new highly reproducible procedure for human breast cancer MCF-7 stem cells (BCSC) isolation and selection was developed by using a combination of hypoxia/hypoglycemia plus taxol and adriamycin for 24h. The BCSC enriched fraction (i) expressed (2-15 times) the typical stemness protein markers CD44+, ALDH1A3 and Oct 3/4; (ii) increased its clonogenicity index (20-times), invasiveness profile (>70%), migration capacity (100%) and ability to form mammospheres, compared to its non-metastatic MCF-7 counterpart. This isolation and selection protocol was successful to obtain stem cell enriched fractions from A549, SiHa and medulloblastoma cells. Since the secretion of HPI/AMF cytokine seems involved in metastasis, the effects of erytrose-4-phosphate (E4P) and 6-phosphogluconate (6PG), potent HPI inhibitors, on the acquisition of the breast stem cell-like phenotype were also evaluated. The presence of E4P during the BCSC selection deterred the development of the stemness phenotype, whereas both extracellular E4P (5-250nM) and 6PG (1µM) as well as siRNA HPI/AMF depressed the BCSC invasiveness ability (>90%), clonogenicity index (>90%) and contents (50-96%) of stemness (CD44, ALDH1A), pluripotency (p38 MAPK, Oct3/4, wnt/ß-catenin) and EMT (SNAIL, MMP-1, vimentin) markers. The cytokine inhibitor repertaxin (10nM) or the anti-IL-8 or anti-TGF-ß monoclonal antibodies (10µg/mL) did not significantly affect the BCSC metastatic phenotype. E4P also diminished (75%) the formation and growth of MCF-7 stem cell mammospheres. These results suggested that E4P by directly interacting with extracellular HPI/AMF may be an effective strategy to deter BCSC growth and progression.
Assuntos
Neoplasias da Mama/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Transição Epitelial-Mesenquimal/genética , Feminino , Gluconatos/administração & dosagem , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Células MCF-7 , Metástase Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Paclitaxel/administração & dosagem , RNA Interferente Pequeno/genética , Fosfatos Açúcares/administração & dosagem , Sulfonamidas/administração & dosagemRESUMO
The accelerated growth of solid tumors leads to episodes of both hypoxia and hypoglycemia (HH) affecting their intermediary metabolism, signal transduction, and transcriptional activity. A previous study showed that normoxia (20% O2 ) plus 24 h hypoglycemia (2.5 mM glucose) increased glycolytic flux whereas oxidative phosphorylation (OxPhos) was unchanged versus normoglycemia in HeLa cells. However, the simultaneous effect of HH on energy metabolism has not been yet examined. Therefore, the effect of hypoxia (0.1-1% O2 ) plus hypoglycemia on the energy metabolism of HeLa cells was analyzed by evaluating protein content and activity, along with fluxes of both glycolysis and OxPhos. Under hypoxia, in which cell growth ceased and OxPhos enzyme activities, ΔΨm and flux were depressed, hypoglycemia did not stimulate glycolytic flux despite increasing H-RAS, p-AMPK, GLUT1, GLUT3, and HKI levels, and further decreasing mitochondrial enzyme content. The impaired mitochondrial function in HH cells correlated with mitophagy activation. The depressed OxPhos and unchanged glycolysis pattern was also observed in quiescent cells from mature multicellular tumor spheroids, suggesting that these inner cell layers are similarly subjected to HH. The principal ATP supplier was glycolysis for HH 2D monolayer and 3D quiescent spheroid cells. Accordingly, the glycolytic inhibitors iodoacetate and gossypol were more effective than mitochondrial inhibitors in decreasing HH-cancer cell viability. Under HH, stem cell-, angiogenic-, and EMT-biomarkers, as well as glycoprotein-P content and invasiveness, were also enhanced. These observations indicate that HH cancer cells develop an attenuated Warburg and pronounced EMT- and invasive-phenotype. J. Cell. Physiol. 232: 1346-1359, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Transição Epitelial-Mesenquimal , Glicólise , Hipoglicemia/patologia , Esferoides Celulares/patologia , Trifosfato de Adenosina/farmacologia , Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Células HeLa , Humanos , Concentração Inibidora 50 , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Invasividade Neoplásica , Oxigênio/farmacologia , Fenótipo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismoRESUMO
Cancer is among the leading causes of death worldwide, and the number of new cases continues to rise. Despite recent advances in diagnosis and therapeutic strategies, millions of cancer-related deaths occur, indicating the need for better therapies and diagnostic strategies. Mitochondria and metabolic alterations have been recognized as important for cancer progression. However, a more precise understanding of how to manipulate mitochondria-related processes for cancer therapy remains to be established. Mitochondria are highly dynamic organelles which continually fuse and divide in response to diverse stimuli. Participation in the aforementioned processes requires a precise regulation at many levels that allows the cell to couple mitochondrial activity to nutrient availability, biosynthetic demands, proliferation rates, and external stimuli. The many functions of these organelles are intimately linked to their morphology. Recent evidence suggests an important link between mitochondrial morphology and disease, including neurodegenerative, inflammatory diseases and cancer. Here, we review recent advances in the understanding of mitochondrial dynamics with a special focus on its relationship to tumor progression.
Assuntos
Mitocôndrias/genética , Dinâmica Mitocondrial/genética , Neoplasias/genética , Humanos , Mitocôndrias/metabolismo , Neoplasias/patologiaRESUMO
BACKGROUND: Exceedingly high therapeutic/experimental doses of metabolic drugs such as oxamate, aminooxyacetate (AOA) and dichloroacetate (DCA) are required to diminish growth, glycolysis and oxidative phosphorylation (OxPhos) of different cancer cells. To identify the mechanisms of action of these drugs on cancer energy metabolism, a systematic analysis of their specificities was undertaken. METHODS: Hepatocarcinoma AS-30D cells were treated with the inhibitors and glycolysis and OxPhos enzyme activities, metabolites and fluxes were analyzed. Kinetic modeling of glycolysis was used to identify the regulatory mechanisms. RESULTS: Oxamate (i) not only inhibited LDH, but also PYK and ENO activities inducing an increase in the cytosolic NAD(P)H, Fru1,6BP and DHAP levels in AS-30D cells; (ii) it slightly inhibited HPI, ALD and Glc6PDH; and (iii) it inhibited pyruvate-driven OxPhos in isolated heart mitochondria. AOA (i) strongly inhibited both AAT and AlaT, and 2-OGDH and glutamate-driven OxPhos; and (ii) moderately affected GAPDH and TPI. DCA slightly affected pyruvate-driven OxPhos and Glc6PDH. Kinetic modeling of cancer glycolysis revealed that oxamate inhibition of LDH, PYK and ENO was insufficient to achieve glycolysis flux inhibition. To do so, HK, HPI, TPI and GAPDH have to be also inhibited by the accumulated Fru1,6BP and DHAP induced by oxamate. CONCLUSION: Oxamate, AOA, and DCA are not specific drugs since they inhibit several enzymes/transporters of the glycolytic and OxPhos pathways through direct interaction or indirect mechanisms. GENERAL SIGNIFICANCE: These data explain why oxamate or AOA, through their multisite inhibitory actions on glycolysis or OxPhos, may be able to decrease the proliferation of cancer cells.
Assuntos
Ácido Amino-Oxiacético/farmacologia , Ácido Dicloroacético/farmacologia , Metabolismo Energético/efeitos dos fármacos , Neoplasias/metabolismo , Ácido Oxâmico/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Simulação por Computador , Fosfato de Di-Hidroxiacetona/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Glicólise/efeitos dos fármacos , Humanos , Cinética , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Modelos Moleculares , NADP/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Ratos Wistar , Sus scrofaRESUMO
BACKGROUND: Ski and SnoN proteins function as transcriptional co-repressors in the TGF-ß pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-ß signalling, malignant transformation, and alterations in cell proliferation. METHODS: We carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-ß, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-ß/Smad-driven gene transcription. RESULTS: Ski and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-ß-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-ß-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-ß-target genes. CONCLUSION: Normal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells. GENERAL SIGNIFICANCE: This work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.
Assuntos
Actinas/química , Citoesqueleto/química , Proteínas de Ligação a DNA/química , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Proto-Oncogênicas/química , Animais , Carcinoma Hepatocelular/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Polimerização , Estabilidade Proteica , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Epithelial-mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2-15 times) of EMT (ß-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (>80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.
Assuntos
Neoplasias da Mama/prevenção & controle , Carcinoma Ductal de Mama/prevenção & controle , Movimento Celular/efeitos dos fármacos , Gluconatos/farmacologia , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Esferoides Celulares/efeitos dos fármacos , Fosfatos Açúcares/farmacologia , Células 3T3 , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/secundário , Proliferação de Células/efeitos dos fármacos , Estudos Transversais , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Glucose-6-Fosfato Isomerase/metabolismo , Humanos , Ácido Láctico/metabolismo , Células MCF-7 , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Fenótipo , Esferoides Celulares/patologiaRESUMO
During multi-cellular tumor spheroid growth, oxygen and nutrient gradients develop inducing specific genetic and metabolic changes in the proliferative and quiescent cellular layers. An integral analysis of proteomics, metabolomics, kinetomics and fluxomics revealed that both proliferative- (PRL) and quiescent-enriched (QS) cellular layers of mature breast tumor MCF-7 multi-cellular spheroids maintained similar glycolytic rates (3-5 nmol/min/10(6) cells), correlating with similar GLUT1, GLUT3, PFK-1, and HKII contents, and HK and LDH activities. Enhanced glycolytic fluxes in both cell layer fractions also correlated with higher HIF-1α content, compared to MCF-7 monolayer cultures. On the contrary, the contents of the mitochondrial proteins GA-K, ND1, COXIV, PDH-E1α, 2-OGDH, SDH and F1-ATP synthase (20 times) and the oxidative phosphorylation (OxPhos) flux (2-times) were higher in PRL vs. QS. Enhanced mitochondrial metabolism in the PRL layers correlated with an increase in the oncogenes h-Ras and c-Myc, and transcription factors p32 and PGC-1α, which are involved in the OxPhos activation. On the other hand, the lower mitochondrial function in QS was associated with an increase in Beclin, LC3B, Bnip3 and LAMP protein levels, indicating active mitophagy and lysosome biosynthesis processes. Although a substantial increase in glycolysis was developed, OxPhos was the predominant ATP supplier in both QS and PRL layers. Therefore, targeted anti-mitochondrial therapy by using oligomycin (IC(50)=11 nM), Casiopeina II-gly (IC(50)=40 nM) or Mitoves (IC(50)=7 nM) was effective to arrest MCF-7 spheroid growth without apparent effect on normal epithelial breast tissue at similar doses; canonical anti-neoplastic drugs such as cisplatin and tamoxifen were significantly less potent.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células , Proteínas Mitocondriais/metabolismo , Mitofagia/efeitos dos fármacos , Oligomicinas/farmacologia , Compostos Organometálicos/farmacologia , Esferoides Celulares/efeitos dos fármacos , Antineoplásicos/farmacologia , Western Blotting , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cobre/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Glicólise/efeitos dos fármacos , Humanos , Técnicas Imunoenzimáticas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteoma/análise , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Desacopladores/farmacologiaRESUMO
Significant efforts have been made for the development of new anticancer drugs (protein kinase or proteasome inhibitors, monoclonal humanized antibodies) with presumably low or negligible side effects and high specificity. However, an in-depth analysis of the side effects of several currently used canonical (platin-based drugs, taxanes, anthracyclines, etoposides, antimetabolites) and new generation anticancer drugs as the first line of clinical treatment reveals significant perturbation of glycolysis and oxidative phosphorylation. Canonical and new generation drug side effects include decreased (1) intracellular ATP levels, (2) glycolytic/mitochondrial enzyme/transporter activities and/or (3) mitochondrial electrical membrane potentials. Furthermore, the anti-proliferative effects of these drugs are markedly attenuated in tumor rho (0) cells, in which functional mitochondria are absent; in addition, several anticancer drugs directly interact with isolated mitochondria affecting their functions. Therefore, several anticancer drugs also target the energy metabolism, and hence, the documented inhibitory effect of anticancer drugs on cancer growth should also be linked to the blocking of ATP supply pathways. These often overlooked effects of canonical and new generation anticancer drugs emphasize the role of energy metabolism in maintaining cancer cells viable and its targeting as a complementary and successful strategy for cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/efeitos adversos , Desenho de Fármacos , Glicólise/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/patologiaRESUMO
Metabolic alterations are recognized as one of the hallmarks of cancer. Among these, alterations in mitochondrial function have been associated with an enhanced production of Reactive Oxygen Species (ROS), which activate ROS-regulated cancer cell signaling pathways. Breast cancer is the main cancer-related cause of death for women globally. It is a heterogeneous disease with subtypes characterized by specific molecular features and patient outcomes. With the purpose of identifying differences in energy metabolism and the oxidative stress management system in non-tumorigenic, estrogen receptor positive (ER+) and triple negative (TN) breast cancer cells, we evaluated ROS production, protein enzyme levels and activities and profiled energy metabolism. We found differences in energetic metabolism and ROS management systems between non-tumorigenic and cancer cells and between ER+ and TN breast cancer cells. Our results indicate a dependence on glycolysis despite different glycolytic ATP levels in all cancer cell lines tested. In addition, our data show that high levels of ROS in TN cells are a result of limited antioxidant capacity in the NADPH producing and GSH systems, mitochondrial dysfunction and non-mitochondrial ROS production, making them more sensitive to GSH synthesis inhibitors. Our data suggest that metabolic and antioxidant profiling of breast cancer will provide important targets for metabolic inhibitors or antioxidant treatments for breast cancer therapy.
RESUMO
Glioblastoma multiforme (GBM) exhibits genetic alterations that induce the deregulation of oncogenic pathways, thus promoting metabolic adaptation. The modulation of metabolic enzyme activities is necessary to generate nucleotides, amino acids, and fatty acids, which provide energy and metabolic intermediates essential for fulfilling the biosynthetic needs of glioma cells. Moreover, the TCA cycle produces intermediates that play important roles in the metabolism of glucose, fatty acids, or non-essential amino acids, and act as signaling molecules associated with the activation of oncogenic pathways, transcriptional changes, and epigenetic modifications. In this review, we aim to explore how dysregulated metabolic enzymes from the TCA cycle and oxidative phosphorylation, along with their metabolites, modulate both catabolic and anabolic metabolic pathways, as well as pro-oncogenic signaling pathways, transcriptional changes, and epigenetic modifications in GBM cells, contributing to the formation, survival, growth, and invasion of glioma cells. Additionally, we discuss promising therapeutic strategies targeting key players in metabolic regulation. Therefore, understanding metabolic reprogramming is necessary to fully comprehend the biology of malignant gliomas and significantly improve patient survival.
RESUMO
Despite abundant evidence correlating T cell CD38 expression and HIV infection pathogenesis, its role as a CD4T cell immunometabolic regulator remains unclear. We find that CD38's extracellular glycohydrolase activity restricts metabolic reprogramming after T cell receptor (TCR)-engaging stimulation in Jurkat T CD4 cells, together with functional responses, while reducing intracellular nicotinamide adenine dinucleotide and nicotinamide mononucleotide concentrations. Selective elimination of CD38's ectoenzyme function licenses them to decrease the oxygen consumption rate/extracellular acidification rate ratio upon TCR signaling and to increase cycling, proliferation, survival, and CD40L induction. Pharmacological inhibition of ecto-CD38 catalytic activity in TM cells from chronic HIV-infected patients rescued TCR-triggered responses, including differentiation and effector functions, while reverting abnormally increased basal glycolysis, cycling, and spontaneous proinflammatory cytokine production. Additionally, ecto-CD38 blockage normalized basal and TCR-induced mitochondrial morphofunctionality, while increasing respiratory capacity in cells from HIV+ patients and healthy individuals. Ectoenzyme CD38's immunometabolic restriction of TCR-involving stimulation is relevant to CD4T cell biology and to the deleterious effects of CD38 overexpression in HIV disease.
Assuntos
ADP-Ribosil Ciclase 1 , Linfócitos T CD4-Positivos , Infecções por HIV , Humanos , ADP-Ribosil Ciclase 1/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Jurkat , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Glicoproteínas de Membrana/metabolismo , Glicólise , Mitocôndrias/metabolismoRESUMO
The effects of α-tocopheryl succinate (α-TOS), α-tocopheryl acetyl ether (α-TEA) and triphenylphosphonium-tagged vitamin E succinate (mitochondrially targeted vitamin E succinate; MitoVES) on energy-related mitochondrial functions were determined in mitochondria isolated from AS-30D hepatoma and rat liver, bovine heart sub-mitochondrial particles (SMPs), and in rodent and human carcinoma cell lines and rat hepatocytes. In isolated mitochondria, MitoVES stimulated basal respiration and ATP hydrolysis, but inhibited net state 3 (ADP-stimulated) respiration and Ca(2+) uptake, by collapsing the membrane potential at low doses (1-10µM). Uncoupled mitochondrial respiration and basal respiration of SMPs were inhibited by the three drugs at concentrations at least one order of magnitude higher and with different efficacy: MitoVES>α-TEA>α-TOS. At high doses (>10µM), the respiratory complex II (CII) was the most sensitive MitoVES target. Acting as an uncoupler at low doses, this agent stimulated total O(2) uptake, collapsed ∆ψ(m), inhibited oxidative phosphorylation and induced ATP depletion in rodent and human cancer cells more potently than in normal rat hepatocytes. These findings revealed that in situ tumor mitochondria are preferred targets of the drug, indicating its clinical relevance.
Assuntos
Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Vitamina E/análogos & derivados , Trifosfato de Adenosina/química , Animais , Cálcio/metabolismo , Bovinos , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/fisiologia , RatosRESUMO
The use of kinetic modeling and metabolic control analysis (MCA) to identify possible therapeutic targets and to investigate the controlling and regulatory mechanisms in cancer glycolysis is here reviewed. The glycolytic pathway has been considered a target to decrease cancer cell growth; however, its occurrence in normal cells makes it difficult to design therapeutic strategies that target this pathway in pathological cells. Notwithstanding, the over-expression of all enzymes and transporters, as well as the expression of isoenzymes with different kinetic and regulatory properties in cancer cells, suggested a different distribution of the control of glycolytic flux than that observed in normal cells. Kinetic models of glycolysis are constructed with enzyme kinetics experimental data, validated with the steady-state metabolite concentrations and glycolytic fluxes; applying MCA, permitted us to identify the steps with the highest control of glycolysis in cancer cells, but low control in normal cells. The cancer glycolysis main controlling steps under several metabolic conditions were: glucose transport, hexokinase and hexose-6-phosphate isomerase (HPI); whereas in normal cells were: the first two and phosphofructokinase-1. HPI is the best therapeutic target because it exerts high control in cancer glycolytic flux, but not in normal cells. Furthermore, kinetic modeling also contributed to identifying new feed-back and feed-forward regulatory loops in cancer cells glycolysis, and to understanding the mode of metabolic action of glycolytic inhibitors. Thus, MCA and metabolic modeling allowed to propose new strategies for inhibiting glycolysis in cancer cells.
Assuntos
Modelos Biológicos , Neoplasias , Humanos , Glicólise , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Hexoquinase/metabolismo , CinéticaRESUMO
Cellular interactions within the bone marrow microenvironment modulate the properties of subsets of leukemic cells leading to the development of drug-resistant phenotypes. The intercellular transfer of proteins and organelles contributes to this process but the set of transferred proteins and their effects in the receiving cells remain unclear. This study aimed to detect the intercellular protein transfer from mouse bone marrow stromal cells (OP9 cell line) to human T-lymphoblasts (CCRF-CEM cell line) using nanoLC-MS/MS-based shotgun proteomics in a 3D co-culture system. After 24 h of co-culture, 1513 and 67 proteins from human and mouse origin, respectively, were identified in CCRF-CEM cells. The presence of mouse proteins in the human cell line, detected by analyzing the differences in amino acid sequences of orthologous peptides, was interpreted as the result of intercellular transfer. The transferred proteins might have contributed to the observed resistance to vincristine, methotrexate, and hydrogen peroxide in the co-cultured leukemic cells. Our results suggest that shotgun proteomic analyses of co-cultured cells from different species could be a simple option to get a preliminary survey of the proteins exchanged among interacting cells.
RESUMO
The incidence of kidney disease is increasing worldwide. Acute kidney injury (AKI) can strongly favor cardio-renal syndrome (CRS) type 3 development. However, the mechanism involved in CRS development is not entirely understood. In this sense, mitochondrial impairment in both organs has become a central axis in CRS physiopathology. This study aimed to elucidate the molecular mechanisms associated with cardiac mitochondrial impairment and its role in CRS development in the folic acid-induced AKI (FA-AKI) model. Our results showed that 48 h after FA-AKI, the administration of N-acetyl-cysteine (NAC), a mitochondrial glutathione regulator, prevented the early increase in inflammatory and cell death markers and oxidative stress in the heart. This was associated with the ability of NAC to protect heart mitochondrial bioenergetics, principally oxidative phosphorylation (OXPHOS) and membrane potential, through complex I activity and the preservation of glutathione balance, thus preventing mitochondrial dynamics shifting to fission and the decreases in mitochondrial biogenesis and mass. Our data show, for the first time, that mitochondrial bioenergetics impairment plays a critical role in the mechanism that leads to heart damage. Furthermore, NAC heart mitochondrial preservation during an AKI event can be a valuable strategy to prevent CRS type 3 development.
RESUMO
Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 µM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 µM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Complexo II de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Vitamina E/administração & dosagem , Animais , Antineoplásicos/farmacologia , Bovinos , Transporte de Elétrons , Humanos , Concentração Inibidora 50 , Células Jurkat , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase , Vitamina E/farmacologiaRESUMO
Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes.