RESUMO
Glycolytic and respiratory fluxes were analyzed in cancer and non-cancer cells. The steady-state fluxes in energy metabolism were used to estimate the contributions of aerobic glycolytic and oxidative phosphorylation (OxPhos) pathways to the cellular ATP supply. The rate of lactate production - corrected for the fraction generated by glutaminolysis - is proposed as the appropriate way to estimate glycolytic flux. In general, the glycolytic rates estimated for cancer cells are higher than those found in non-cancer cells, as originally observed by Otto Warburg. The rate of basal or endogenous cellular O2 consumption corrected for non-ATP synthesizing O2 consumption, measured after inhibition by oligomycin (a specific, potent and permeable ATP synthase inhibitor), has been proposed as the appropriate way to estimate mitochondrial ATP synthesis-linked O2 flux or net OxPhos flux in living cells. Detecting non-negligible oligomycin-sensitive O2 consumption rates in cancer cells has revealed that the mitochondrial function is not impaired, as claimed by the Warburg effect. Furthermore, when calculating the relative contributions to cellular ATP supply, under a variety of environmental conditions and for different types of cancer cells, it was found that OxPhos pathway was the main ATP provider over glycolysis. Hence, OxPhos pathway targeting can be successfully used to block in cancer cells ATP-dependent processes such as migration. These observations may guide the re-design of novel targeted therapies.
Assuntos
Trifosfato de Adenosina , Neoplasias , Trifosfato de Adenosina/metabolismo , Metabolismo Energético , Glicólise/fisiologia , Fosforilação Oxidativa , Ciclo do Ácido CítricoRESUMO
Over the last decade, three major advances have contributed in improving the response rates against cancer including, immunotherapy; greater understanding of the molecular, biochemical, and cellular mechanisms in carcinogenesis thereby providing drug targets; and identification of reliable biomarkers for early detection to facilitate the earlier stage treatment of disease. However, no single universal cancer cure has yet been found, although combinations from the above areas have steadily improved survival outcomes. Hence, chemotherapy remains a key component in the oncologist's arsenal for cancer therapy, despite frequent development of drug resistance and more aggressive cancers with onset of advanced stage metastases. The focus here is to explore the repurposing of old drugs that cause pro-oxidative overload to overcome onset of resistance to chemotherapy and enhance chemotherapeutic responses, particularly against metastatic cancer. Excellent examples of US Food and Drug Administration approved drugs suitable for repurposing are the potent and specific thioreductase inhibitor auranofin and the nonsteroidal anti-inflammatory drug, celecoxib. Recently, both drugs were shown to selectively target and kill metastatic cancer cells and cancer stem cells (CSCs), predominantly by promoting excessive mitochondrial reactive oxygen species. Thus, targeting intracellular redox systems of advanced stage metastatic cancer cells and CSCs can promote an overload of pro-oxidative stress to activate the intrinsic pathway for programmed cell death. It is envisaged that more clinical studies will incorporate longer term use of repurposed drugs, such as auranofin or celecoxib, to target redox systems in cancer cells as part of common practice postcancer diagnosis, providing enhanced chemotherapeutic responses and increased cancer survival.
Assuntos
Reposicionamento de Medicamentos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Oxidantes/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , OxirreduçãoRESUMO
Mutations in p53 are strongly associated with several highly malignant cancer phenotypes but its role in regulating energy metabolism has not been completely elucidated. The effect on glycolysis and oxidative phosphorylation (OxPhos) of mutant p53R248Q overexpression in HeLa cells (HeLa-M) was analyzed and compared with cells overexpressing wild-type p53 (HeLa-H) and nontransfected cells containing negligible p53 levels (HeLa-L). p53 R248Q overexpression induced early cell detachment during in vitro growth; however, detached HeLa-M cells showed high viability, shorter generation time and significant diminution in the adhesion proteins E-cadherin and ß-catenin versus HeLa-H and HeLa-L cells. Under normoxia, a lower growth rate of attached HeLa-M cells correlated with decreased levels of proliferating cell nuclear antigen (PCNA), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), adenosine monophosphate-activated protein kinase (AMPK), mitochondrial proteins (20-80%) and OxPhos flux (69 ± 12%). On the contrary, HeLa-M also showed increased contents of CDKN1A, nuclear factor κB (NF-κB), c-MYC, hypoxia-inducible factor 1-α (HIF-1α; 1-4 times), glycolytic HIF-1α targets (2-4 times), and glycolysis flux (2-fold) versus HeLa-H. In consequence, glycolysis provided ~70% of the cellular adenosine triphosphate (ATP) in HeLa-M cells under normoxia whereas, OxPhos predominated (65-82%) in HeLa-H and HeLa-L cells. Pifithrin-α, a specific p53 inhibitor, did not alter the p53 R248Q target protein contents and OxPhos and glycolytic fluxes, and a poor HIF-1α-p53 R248Q interaction was attained, in HeLa-M cells. These observations suggested that p53 R248Q deficiently interacted with pifithrin-α and HIF-1α. Therefore, lower mitochondrial biogenesis, deficient HIF-1α/mutant p53 interaction, and development of a pseudohypoxic state under normoxia were the apparent biochemical mechanisms underlying glycolysis activation and OxPhos downregulation in HeLa-M cells.
Assuntos
Glicólise , Mutação , Fosforilação Oxidativa , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Proliferação de Células , Feminino , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Biogênese de Organelas , Hipóxia Tumoral , Microambiente Tumoral , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
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
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
To establish alternative targeted therapies against triple negative (TN) breast cancer, the energy metabolism and the sensitivity of cell growth, migration, and invasiveness toward metabolic, canonical, and NSAID inhibitors were analyzed in MDA-MB-231 and MDA-MB-468, two TN metastatic breast cancer cell lines, under both normoxia (21% O2) and hypoxia (0.1% O2). For comparative purposes, the analysis was also carried out in the less-metastatic breast MCF-7 cancer cells. Under normoxia, oxidative phosphorylation (OxPhos) was significantly higher (2-times) in MDA-MB-468 than in MDA-MB-231 and MCF-7, whereas their glycolytic fluxes and OxPhos and glycolytic protein contents were all similar. TN cancer cell lines mainly depended on OxPhos (62-75%), whereas MCF-7 cells equally depended on both pathways for ATP supply. Hypoxia for 24 h promoted a significant increase (>20 times) in the glycolytic transcriptional master factor HIF1-α in its target proteins GLUT-1, HKI and II, and LDH-A (2-4 times) as well as in the glycolytic flux (1.3-2 times) vs normoxia in MDA-MB-468, MDA-MB-231, and MCF-7. On the contrary, hypoxia decreased (15-60%) the contents of COXIV, 2OGDH, ND1, and ATP synthase as well as the OxPhos flux (50-75%), correlating with a high mitophagy level in the three cell lines. Under hypoxia, the three cancer cell lines mainly depended on glycolysis (70-80%). Anti-mitochondrial drugs (oligomycin, casiopeina II-gly, and methoxy-TEA) and celecoxib, at doses used to block OxPhos, significantly decreased TN cancer cell proliferation (IC50 = 2-20 µM), migration capacity (10-90%), and invasiveness (25-65%). The present data support the use of mitochondrially targeted inhibitors for the treatment of TN breast carcinoma.
Assuntos
Antineoplásicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Células 3T3 , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
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
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
Free-living microorganisms may become suitable models for removal of heavy metals from polluted water bodies, sediments, and soils by using and enhancing their metal accumulating abilities. The available research data indicate that protists of the genus Euglena are a highly promising group of microorganisms to be used in bio-remediation of heavy metal-polluted aerobic and anaerobic acidic aquatic environments. This chapter analyzes the variety of biochemical mechanisms evolved in E. gracilis to resist, accumulate and remove heavy metals from the environment, being the most relevant those involving (1) adsorption to the external cell pellicle; (2) intracellular binding by glutathione and glutathione polymers, and their further compartmentalization as heavy metal-complexes into chloroplasts and mitochondria; (3) polyphosphate biosynthesis; and (4) secretion of organic acids. The available data at the transcriptional, kinetic and metabolic levels on these metabolic/cellular processes are herein reviewed and analyzed to provide mechanistic basis for developing genetically engineered Euglena cells that may have a greater removal and accumulating capacity for bioremediation and recycling of heavy metals.
Assuntos
Resistência a Medicamentos/fisiologia , Euglena/fisiologia , Metais Pesados/metabolismo , Biodegradação AmbientalRESUMO
The role of p53 as modulator of OxPhos and glycolysis was analyzed in HeLa-L (cells containing negligible p53 protein levels) and HeLa-H (p53-overexpressing) human cervix cancer cells under normoxia and hypoxia. In normoxia, functional p53, mitochondrial enzyme contents, mitochondrial electrical potential (ΔΨm) and OxPhos flux increased in HeLa-H vs. HeLa-L cells; whereas their glycolytic enzyme contents and glycolysis flux were unchanged. OxPhos provided more than 70% of the cellular ATP and proliferation was abolished by anti-mitochondrial drugs in HeLa-H cells. In hypoxia, both cell proliferations were suppressed, but HeLa-H cells exhibited a significant decrease in OxPhos protein contents, ΔΨm and OxPhos flux. Although glycolytic function was also diminished vs. HeLa-L cells in hypoxia, glycolysis provided more than 60% of cellular ATP in HeLa-H cells. The energy metabolism phenotype of HeLa-H cells was reverted to that of HeLa-L cells by incubating with pifithrin-α, a p53-inhibitor. In normoxia, the energy metabolism phenotype of breast cancer MCF-7 cells was similar to that of HeLa-H cells, whereas p53shRNAMCF-7 cells resembled the HeLa-L cell phenotype. In hypoxia, autophagy proteins and lysosomes contents increased 2-5 times in HeLa-H cells suggesting mitophagy activation. These results indicated that under normoxia p53 up-regulated OxPhos without affecting glycolysis, whereas under hypoxia, p53 down-regulated both OxPhos (severely) and glycolysis (weakly). These p53 effects appeared mediated by the formation of p53-HIF-1α complexes. Therefore, p53 exerts a dual and contrasting regulatory role on cancer energy metabolism, depending on the O2level.
Assuntos
Neoplasias da Mama/metabolismo , Metabolismo Energético , Proteína Supressora de Tumor p53/fisiologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias da Mama/patologia , Divisão Celular , Hipóxia Celular , Feminino , Células HeLa , Humanos , Células MCF-7 , Neoplasias do Colo do Útero/patologiaRESUMO
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
Accelerated aerobic glycolysis is one of the main metabolic alterations in cancer, associated with malignancy and tumor growth. Although glycolysis is one of the most studied properties of tumor cells, recent studies demonstrate that oxidative phosphorylation (OxPhos) is the main ATP provider for the growth and development of cancer. In this last regard, the levels of mRNA and protein of OxPhos enzymes and transporters (including glutaminolysis, acetate and ketone bodies catabolism, free fatty acid ß-oxidation, Krebs Cycle, respiratory chain, phosphorylating system- ATP synthase, ATP/ADP translocator, Pi carrier) are altered in tumors and cancer cells in comparison to healthy tissues and organs, and non-cancer cells. Both energy metabolism pathways are tightly regulated by transcriptional factors, oncogenes, and tumor-suppressor genes, all of which dictate their protein levels depending on the micro-environmental conditions and the type of cancer cell, favoring cancer cell adaptation and growth. In the present review paper, variation in the mRNA and protein levels as well as in the enzyme/ transporter activities of the OxPhos machinery is analyzed. An integral omics approach to mitochondrial energy metabolism pathways may allow for identifying their use as suitable, reliable biomarkers for early detection of cancer development and metastasis, and for envisioned novel, alternative therapies.
Assuntos
Biomarcadores Tumorais , Proteínas Mitocondriais , Neoplasias , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/tratamento farmacológico , Biomarcadores Tumorais/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Metabolismo Energético , Animais , Metástase NeoplásicaRESUMO
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
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.
Assuntos
Glicólise/fisiologia , Modelos Teóricos , Neoplasias/metabolismo , Transporte Biológico/fisiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Metabolismo Energético/fisiologia , Ativação Enzimática/fisiologia , Glucose/farmacocinética , Células HeLa , Hexoquinase/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Modelos Biológicos , Neoplasias/enzimologia , Neoplasias/patologia , Concentração Osmolar , Fosfofrutoquinase-1/metabolismo , Células Tumorais CultivadasRESUMO
Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.
Assuntos
Neoplasias/enzimologia , Neoplasias/genética , Fosfofrutoquinase-1/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , DNA Complementar/genética , Ativação Enzimática , Feminino , Células HeLa , Humanos , Cinética , Fígado/enzimologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/genética , Miocárdio/enzimologia , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1 Hepática/genética , Fosfofrutoquinase-1 Hepática/metabolismo , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Fosfofrutoquinase-1 Tipo C/genética , Fosfofrutoquinase-1 Tipo C/metabolismo , Polimorfismo Genético , Ratos , Ratos Wistar , Especificidade por Substrato , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/genéticaRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive of the primary brain tumors, with a grim prognosis despite intensive treatment. In the past decades, progress in research has not significantly increased overall survival rate. METHODS: The in vitro antineoplastic effect and mechanism of action of Casiopeina III-ia (Cas III-ia), a copper compound, on rat malignant glioma C6 cells was investigated. RESULTS: Cas III-ia significantly inhibited cell proliferation, inducing autophagy and apoptosis, which correlated with the formation of autophagic vacuoles, overexpression of LC3, Beclin 1, Atg 7, Bax and Bid proteins. A decrease was detected in the mitochondrial membrane potential and in the activity of caspase 3 and 8, together with the generation of intracellular reactive oxygen species (ROS) and increased activity of c-jun NH(2)-terminal kinase (JNK). The presence of 3-methyladenine (as selective autophagy inhibitor) increased the antineoplastic effect of Cas III-ia, while Z-VAD-FMK only showed partial protection from the antineoplastic effect induced by Cas III-ia, and ROS antioxidants (N-acetylcysteine) decreased apoptosis, autophagy and JNK activity. Moreover, the JNK -specific inhibitor SP600125 prevented Cas III-ia-induced cell death. CONCLUSIONS: Our data suggest that Cas III-ia induces cell death by autophagy and apoptosis, in part due to the activation of ROS -dependent JNK signaling. These findings support further studies of Cas III-ia as candidate for treatment of human malignant glioma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Glioma/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Compostos Organometálicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Autofagia/efeitos dos fármacos , Catalase/metabolismo , Linhagem Celular Tumoral , Cobre , Ativação Enzimática/efeitos dos fármacos , Humanos , Ratos , Superóxido Dismutase/metabolismoRESUMO
The copper-based drug Casiopeina II-gly (CasII-gly) shows potent antineoplastic effect and diminishes mitochondrial metabolism on several human and rodent malignant tumors. To elucidate whether CasII-gly also affects glycolysis, (a) the flux through the complete pathway and the initial segment and (b) the activities of several glycolytic enzymes of AS-30D hepatocarcinoma cells were determined. CasII-gly (IC50 = 0.74-6.7 µM) was more effective to inhibit 24-72 h growth of several human carcinomas than 3-bromopyruvate (3BrPyr) (IC50 = 45-100 µM) with no apparent effect on normal human-proliferating lymphocytes and HUVECs. In short-term 60-min experiments, CasII-gly increased tumor cell lactate production and glycogen breakdown. CasII-gly was 1.3-21 times more potent than 3BrPyr and cisplatin to inhibit tumor HK. As CasII-gly inhibited the soluble and mitochondrial HK activities and the flux through the HK-TPI glycolytic segment, whereas PFK-1, GAPDH, PGK, PYK activities and HPI-TPI segment flux were not affected, the data suggested glycogenolysis activation induced by HK inhibition. Accordingly, glycogen-depleted as well as oligomycin-treated cancer cells became more sensitive to CasII-gly. The inhibition time-course of HK by CasII-gly was slower than that of OxPhos in AS-30D cells, indicating that glycolytic toxicity was secondary to mitochondria, the primary CasII-gly target. In long-term 24-h experiments with HeLa cells, 5 µM CasII-gly inhibited OxPhos (80%), glycolysis (40%), and HK (42%). The present data indicated that CasII-gly is an effective multisite anticancer drug simultaneously targeting mitochondria and glycolysis.