RESUMEN
The cananga tree alkaloid sampangine (1) has been extensively investigated for its antimicrobial and antitumor potential. Mechanistic studies have linked its biological activities to the reduction of cellular oxygen, the induction of reactive oxygen species (ROS), and alterations in heme biosynthesis. Based on the yeast gene deletion library screening results that indicated mitochondrial gene deletions enhanced the sensitivity to 1, the effects of 1 on cellular respiration were examined. Sampangine increased oxygen consumption rates in both yeast and human tumor cells. Mechanistic investigation indicated that 1 may have a modest uncoupling effect, but predominately acts by increasing oxygen consumption independent of mitochondrial complex IV. Sampangine thus appears to undergo redox cycling that may involve respiratory chain-dependent reduction to a semi-iminoquinone followed by oxidation and consequent superoxide production. Relatively high concentrations of 1 showed significant neurotoxicity in studies conducted with rat cerebellar granule neurons, indicating that sampangine use may be associated with potential neurotoxicity.
Asunto(s)
Alcaloides/farmacología , Antiinfecciosos/farmacología , Antineoplásicos Fitogénicos/farmacología , Quinonas/farmacología , Animales , Benzoquinonas , Ciclo Celular/efectos de los fármacos , División Celular , Respiración de la Célula/efectos de los fármacos , Transporte de Electrón , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Mitocondrias/metabolismo , Estructura Molecular , Naftiridinas , Oxidación-Reducción , Oxígeno , Ratas , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae , Superóxidos/metabolismoRESUMEN
The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-D-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Lípidos/farmacología , Neovascularización Patológica/tratamiento farmacológico , Tiazoles/farmacología , Inhibidores de la Angiogénesis/administración & dosificación , Neoplasias de la Mama/patología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Complejo I de Transporte de Electrón/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Femenino , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Concentración 50 Inhibidora , Lípidos/administración & dosificación , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazoles/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.
Asunto(s)
Caulophyllum/química , Respiración de la Célula/efectos de los fármacos , Suplementos Dietéticos/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ácido Oleanólico/análogos & derivados , Saponinas/toxicidad , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Ácido Oleanólico/química , Ácido Oleanólico/toxicidad , Fitoterapia , Saponinas/química , Estados UnidosRESUMEN
Bioassay-guided isolation and subsequent structure elucidation of a Bael tree Aegle marmelos lipid extract yielded two unstable acylated geranyloxycoumarin mixtures (1-2), six geranyloxycoumarins (3-8), (+)-9'-isovaleroxylariciresinol (9), and dehydromarmeline (10). In a T47D cell-based reporter assay, 1 and 2 potently inhibited hypoxia-induced HIF-1 activation (IC50 values 0.18 and 1.10 µgmL(-1), respectively). Insufficient material and chemical instability prevented full delineation of the fatty acyl side chain olefin substitution patterns in 1 and 2. Therefore, five fatty acyl geranyloxycoumarin ester derivatives (11-15) were prepared from marmin (3) and commercial fatty acyl chlorides by semisynthesis. The unsaturated C-6' linoleic acid ester derivative 14 that was structurally most similar to 1 and 2, inhibited HIF-1 activation with comparable potency (IC50 0.92 µM). The octanoyl (11) and undecanoyl (12) ester derivatives also suppressed HIF-1 activation (IC50 values 3.1 and 0.87 µM, respectively). Mechanistic studies revealed that these geranyloxycoumarin derivatives disrupt mitochondrial respiration, primarily at complex I. Thus, these compounds may inhibit HIF-1 activation by suppressing mitochondria-mediated hypoxic signaling. One surprising observation was that, while less potent, the purported cancer chemopreventive agent auraptene (8) was found to act as a mitochondrial poison that disrupts HIF-1 signaling in tumors.
Asunto(s)
Aegle/química , Cumarinas/química , Cumarinas/toxicidad , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Mitocondrias/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/toxicidad , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/toxicidad , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cumarinas/aislamiento & purificación , Cumarinas/farmacología , Suplementos Dietéticos/efectos adversos , Suplementos Dietéticos/análisis , Femenino , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Mitocondrias/metabolismo , Mitocondrias/patología , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacologíaRESUMEN
Gene expression signatures associated with breast cancer metastases suggest that metabolic re-wiring is important for metastatic growth in lungs, bones, and other organs. However, since pathway fluxes depend on additional factors such as ATP demand, allosteric effects, and post-translational modification, flux analysis is necessary to conclusively establish phenotypes. In this study, the metabolic phenotypes of breast cancer cell lines with low (T47D) or high (MDA-MB-231) metastatic potential, as well as lung (LM)- and bone (BoM)-homing lines derived from MDA-MB-231 cells, were assessed by 13C metabolite labeling from [1,2-13C] glucose or [5-13C] glutamine and the rates of nutrient and oxygen consumption and lactate production. MDA-MB-231 and T47D cells produced 55 and 63%, respectively, of ATP from oxidative phosphorylation, whereas LM and BoM cells were more glycolytic, deriving only 20-25% of their ATP from mitochondria. ATP demand by BoM and LM cells was approximately half the rate of the parent cells. Of the anabolic fluxes assessed, nucleotide synthesis was the major ATP consumer for all cell lines. Glycolytic NADH production by LM cells exceeded the rate at which it could be oxidized by mitochondria, suggesting that the malate-aspartate shuttle was not involved in re-oxidation of these reducing equivalents. Serine synthesis was undetectable in MDA-MB-231 cells, whereas 3-5% of glucose was shunted to serine by LM and BoM lines. Proliferation rates of T47D, BoM, and LM lines tightly correlated with their respiration-normalized NADPH production rates. In contrast, MDA-MB-231 cells produced NADPH and GSH at higher rates, suggesting this line is more oxidatively stressed. Approximately half to two-thirds of NADPH produced by T47D, MDA-MB-231, and BoM cells was from the oxidative PPP, whereas the majority in LM cells was from the folate cycle. All four cell lines used the non-oxidative PPP to produce pentose phosphates, although this was most prominent for LM cells. Taken together, the metabolic phenotypes of LM and BoM lines differed from the parent line and from each other, supporting the metabolic re-wiring hypothesis as a feature of metastasis to lung and bone.
RESUMEN
Tumor cells exhibit enhanced glucose consumption and lactate production even when supplied with adequate oxygen (a phenomenon known as the Warburg effect, or aerobic glycolysis). Pharmacological inhibition of aerobic glycolysis represents a potential tumor-selective approach that targets the metabolic differences between normal and malignant tissues. Human breast tumor MDA-MB-231 cells were used to develop an assay system to discover natural product-based glycolysis inhibitors. The assay employed was based on hypersensitivity to glycolytic inhibition in tumor cells treated with the mitochondrial electron transport inhibitor rotenone. Under such conditions, ATP supply, and hence cell viability, depends exclusively on glycolysis. This assay system was used to evaluate 10648 plant and marine organism extracts from the U.S. National Cancer Institute's Open Repository. Bioassay-guided isolation of an active Moronobea coccinea extract yielded the new bis-geranylacylphloroglucinol derivative moronone (1). Compound 1 exhibited enhanced antiproliferative/cytotoxic activity in the presence of rotenone-imposed metabolic stress on tumor cells. Surprisingly, mechanistic studies revealed that 1 did not inhibit glycolysis, but rather functions as a protonophore that dissipates the mitochondrial proton gradient. In the presence of rotenone, tumor cells may be hypersensitive to protonophores due to increased ATP utilization by the ATP synthase.
Asunto(s)
Clusiaceae/química , Glucólisis/efectos de los fármacos , Floroglucinol/análogos & derivados , Floroglucinol/aislamiento & purificación , Floroglucinol/farmacología , Supervivencia Celular/efectos de los fármacos , Femenino , Glucosa/metabolismo , Humanos , Estructura Molecular , Neoplasias/metabolismo , Floroglucinol/química , Rotenona/farmacologíaRESUMEN
The organic extract of a marine sponge, Petrosia alfiani, selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1-7) including three new compounds: 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1-7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), which possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC(50) value of 0.2 µM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Poríferos/química , Quinonas/aislamiento & purificación , Quinonas/farmacología , Animales , Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Concentración 50 Inhibidora , Quelantes del Hierro/farmacología , Biología Marina , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estructura Molecular , Quinonas/químicaRESUMEN
Mitochondrial outer membrane Bax oligomers are critical for cytochrome c release, but the role of resident mitochondrial proteins in this process remains unclear. Membrane-associated Bax has primarily been studied using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as the solubilizing agent, as it does not induce conformational artifacts, although recent evidence indicates it may have other artifactual effects. The objective of this study was to investigate digitonin as an alternative detergent to assess Bax oligomeric state, and possible interaction with voltage-dependent anion channel (VDAC)1 in cerebellar granule neurons. VDAC1 co-immunoprecipitated with Bax in digitonin extracts from healthy and apoptotic neurons. Two-dimensional blue native-SDS-PAGE revealed five Bax and VDAC1 oligomers having similar masses from 120 to 500 kDa. The levels of two VDAC1 oligomers in Bax 1D1 immunodepleted extracts negatively correlated with levels of co-precipitated VDAC1, indicating the co-precipitated VDAC1 was derived from these oligomers. Immunodepletion with the 6A7 antibody modestly reduced the levels of Bax oligomers from apoptotic but not healthy neurons. A sixth 170 kDa oligomer containing exclusively 6A7 Bax and no VDAC1 was identified after apoptosis induction. CHAPS failed to solubilize VDAC1, and additionally yielded no distinct oligomers. We conclude that digitonin is a potentially useful detergent preserving Bax-VDAC1 interactions that may be disrupted with CHAPS.
Asunto(s)
Corteza Cerebelosa/química , Gránulos Citoplasmáticos/química , Digitonina/farmacología , Neuronas/química , Canal Aniónico 1 Dependiente del Voltaje/química , Proteína X Asociada a bcl-2/química , Animales , Animales Recién Nacidos , Corteza Cerebelosa/citología , Corteza Cerebelosa/metabolismo , Ácidos Cólicos/farmacología , Gránulos Citoplasmáticos/metabolismo , Neuronas/clasificación , Neuronas/metabolismo , Cultivo Primario de Células , Ratas , Ratas Wistar , Solubilidad , Canal Aniónico 1 Dependiente del Voltaje/fisiología , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
In an effort to identify natural product-based molecular-targeted antitumor agents, mammea-type coumarins from the tropical/subtropical plant Mammea americana were found to inhibit the activation of HIF-1 (hypoxia-inducible factor-1) in human breast and prostate tumor cells. In addition to the recently reported mammea E/BB (15), bioassay-guided fractionation of the active extract yielded 14 mammea-type coumarins including three new compounds, mammea F/BB (1), mammea F/BA (2), and mammea C/AA (3). The absolute configuration of C-1' in 1 was determined by the modified Mosher's method on a methylated derivative. These coumarins were evaluated for their effects on mitochondrial respiration, HIF-1 signaling, and tumor cell proliferation/viability. Acetylation of 1 afforded a triacetoxylated product (A-2) that inhibited HIF-1 activation with increased potency in both T47D (IC(50) 0.83 µM for hypoxia-induced) and PC-3 cells (IC(50) 0.94 µM for hypoxia-induced). Coumarins possessing a 6-prenyl-8-(3-methyloxobutyl) substituent pattern exhibited enhanced HIF-1 inhibitory effects. The O-methylated derivatives were less active at inhibiting HIF-1 and suppressing cell proliferation/viability. Mechanistic studies indicate that these compounds act as anionic protonophores that potently uncouple mitochondrial electron transport and disrupt hypoxic signaling.
Asunto(s)
Antineoplásicos Fitogénicos , Respiración de la Célula/efectos de los fármacos , Cumarinas , Factor 1 Inducible por Hipoxia/efectos de los fármacos , Mammea/química , Algoritmos , Antineoplásicos Fitogénicos/síntesis química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Cumarinas/síntesis química , Cumarinas/química , Cumarinas/aislamiento & purificación , Cumarinas/farmacología , Dominica , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Estructura Molecular , Corteza de la Planta/química , Prenilación , Relación Estructura-ActividadRESUMEN
A natural product chemistry-based approach was applied to discover small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1). A Petrosaspongia mycofijiensis marine sponge extract yielded mycothiazole (1), a solid tumor selective compound with no known mechanism for its cell line-dependent cytotoxic activity. Compound 1 inhibited hypoxic HIF-1 signaling in tumor cells (IC(50) 1nM) that correlated with the suppression of hypoxia-stimulated tumor angiogenesis in vitro. However, 1 exhibited pronounced neurotoxicity in vitro. Mechanistic studies revealed that 1 selectively suppresses mitochondrial respiration at complex I (NADH-ubiquinone oxidoreductase). Unlike rotenone, MPP(+), annonaceous acetogenins, piericidin A, and other complex I inhibitors, mycothiazole is a mixed polyketide/peptide-derived compound with a central thiazole moiety. The exquisite potency and structural novelty of 1 suggest that it may serve as a valuable molecular probe for mitochondrial biology and HIF-mediated hypoxic signaling.
Asunto(s)
Complejo I de Transporte de Electrón/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Poríferos/química , Tiazoles/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Complejo I de Transporte de Electrón/metabolismo , Inhibidores Enzimáticos/aislamiento & purificación , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neuronas/efectos de los fármacos , Ratas , Tiazoles/aislamiento & purificaciónRESUMEN
The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC(50) values of 0.96 and 0.89 µM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 µM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlie their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines.
Asunto(s)
Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Cumarinas/aislamiento & purificación , Cumarinas/farmacología , Factor 1 Inducible por Hipoxia/efectos de los fármacos , Mammea/química , Mitocondrias/efectos de los fármacos , Corteza de la Planta , Animales , Antineoplásicos Fitogénicos/química , Cumarinas/química , Modelos Animales de Enfermedad , Dominica , Transporte de Electrón , Femenino , Humanos , Mitocondrias/metabolismo , Estructura Molecular , Corteza de la Planta/química , Prenilación , Factores de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
Products that contain twig extracts of pawpaw (Asimina triloba) are widely consumed anticancer alternative medicines. Pawpaw crude extract (CE) and purified acetogenins inhibited hypoxia-inducible factor-1 (HIF-1)-mediated hypoxic signaling pathways in tumor cells. In T47D cells, pawpaw CE and the acetogenins 10-hydroxyglaucanetin (1), annonacin (2), and annonacin A (3) inhibited hypoxia-induced HIF-1 activation with IC(50) values of 0.02 microg/mL, 12 nM, 13 nM, and 31 nM, respectively. This inhibition correlates with the suppression of the hypoxic induction of HIF-1 target genes VEGF and GLUT-1. The induction of secreted VEGF protein represents a key event in hypoxia-induced tumor angiogenesis. Both the extract and the purified acetogenins blocked the angiogenesis-stimulating activity of hypoxic T47D cells in vitro. Pawpaw extract and acetogenins inhibited HIF-1 activation by blocking the hypoxic induction of nuclear HIF-1alpha protein. The inhibition of HIF-1 activation was associated with the suppression of mitochondrial respiration at complex I. Thus, the inhibition of HIF-1 activation and hypoxic tumor angiogenesis constitutes a novel mechanism of action for these anticancer alternative medicines.
Asunto(s)
Acetogeninas/aislamiento & purificación , Acetogeninas/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Asimina/química , Transportador de Glucosa de Tipo 1/efectos de los fármacos , Factor 1 Inducible por Hipoxia/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Plantas Medicinales/química , Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Acetogeninas/química , Antineoplásicos Fitogénicos/química , Terapias Complementarias , Ensayos de Selección de Medicamentos Antitumorales , Transportador de Glucosa de Tipo 1/análisis , Transportador de Glucosa de Tipo 1/genética , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Factores de Crecimiento Endotelial Vascular/análisis , Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 microM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 microM) blocked the induction of HIF-1alpha protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1alpha protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.
Asunto(s)
Caulerpa/química , Factor 1 Inducible por Hipoxia/efectos de los fármacos , Indoles/farmacología , Colorantes/química , Colorantes/farmacología , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Femenino , Humanos , Indoles/química , Indoles/aislamiento & purificación , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estructura Molecular , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The lipid extract of the marine sponge Mycale sp. inhibited the activation of hypoxia-inducible factor-1 (HIF-1) in a human breast tumor T47D cell-based reporter assay. Bioassay-guided isolation and structure elucidation yielded 18 new lipophilic 2,5-disubstituted pyrroles and eight structurally related known compounds. The active compounds inhibited hypoxia-induced HIF activation with moderate potency (IC50 values <10 microM). Mechanistic studies revealed that the active compounds suppressed mitochondrial respiration by blocking NADH-ubiquinone oxidoreductase (complex I) at concentrations that inhibited HIF-1 activation. Under hypoxic conditions, reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1alpha protein induction and activation. By inhibiting electron transport (or delivery) to complex III under hypoxic conditions, lipophilic Mycale pyrroles appear to disrupt mitochondrial ROS-regulated HIF-1 signaling.
Asunto(s)
Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Factor 1 Inducible por Hipoxia/metabolismo , Mitocondrias/metabolismo , Poríferos/química , Pirroles/aislamiento & purificación , Pirroles/farmacología , Animales , Respiración de la Célula/efectos de los fármacos , Femenino , Humanos , Biología Marina , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Palau , Pirroles/químicaRESUMEN
Reduced glycolytic and mitochondrial respiration rates are common features of apoptosis that may reflect key events contributing to cell death. However, it is unclear to what extent the rate changes can be explained by direct alterations in the kinetics of the participating reactions, as changes in the concentrations of intermediates also affect reaction rates. Direct kinetic changes can be identified, ranked, and compared to the indirect effects mediated by the intermediates using top-down control analysis. Flux changes that are explained primarily by direct effects are likely to be prime targets of the pathways that signal death, and thus important contributors to apoptosis. Control analysis concepts relevant to identifying such effects are reviewed. Metabolic flux measurements are essential for this approach, but can be technically difficult, particularly when using adherent cells such as neurons. A simple method is described that renders such measurements feasible.
Asunto(s)
Apoptosis , Metabolismo Energético , Neuronas/metabolismo , Biología de Sistemas , Animales , Humanos , Metabolómica , Modelos BiológicosRESUMEN
A natural product chemistry-based approach was employed to discover small-molecule inhibitors of the important tumor-selective molecular target hypoxia-inducible factor-1 (HIF-1). Bioassay-guided isolation of an active lipid extract of a Saipan collection of the marine sponge Lendenfeldia sp. afforded the terpene-derived furanolipid furospongolide as the primary inhibitor of hypoxia-induced HIF-1 activation (IC(50) 2.9 µM, T47D breast tumor cells). The active component of the extract also contained one new cytotoxic scalarane sesterterpene and two previously reported scalaranes. Furospongolide blocked the induction of the downstream HIF-1 target secreted vascular endothelial growth factor (VEGF) and was shown to suppress HIF-1 activation by inhibiting the hypoxic induction of HIF-1α protein. Mechanistic studies indicate that furospongolide inhibits HIF-1 activity primarily by suppressing tumor cell respiration via the blockade of NADH-ubiquinone oxidoreductase (complex I)-mediated mitochondrial electron transfer.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Furanos/aislamiento & purificación , Furanos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Poríferos/química , Animales , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Furanos/química , Humanos , Biología Marina , Estructura Molecular , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacosRESUMEN
Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.
Asunto(s)
Mitocondrias/metabolismo , Mitocondrias/patología , Modelos Biológicos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Animales , HumanosRESUMEN
A hexose phosphate recycling model previously developed to infer fluxes through the major glucose consuming pathways in cultured cerebellar granule neurons (CGNs) from neonatal rats metabolizing [1,2-13C2]glucose was revised by considering reverse flux through the non-oxidative pentose phosphate pathway (PPP) and symmetrical succinate oxidation within the tricarboxylic acid (TCA) cycle. The model adjusts three flux ratios to effect 13C distribution in the hexose, pentose, and triose phosphate pools, and in TCA cycle malate to minimize the error between predicted and measured 13C labeling in exported lactate (i.e., unlabeled, single-, double-, and triple-labeled; M, M1, M2, and M3, respectively). Inclusion of reverse non-oxidative PPP flux substantially increased the number of calculations but ultimately had relatively minor effects on the labeling of glycolytic metabolites. From the error-minimized solution in which the predicted M-M3 lactate differed by 0.49% from that measured by liquid chromatography-triple quadrupole mass spectrometry, the neurons exhibited negligible forward non-oxidative PPP flux. Thus, no glucose was used by the pentose cycle despite explicit consideration of hexose phosphate recycling. Mitochondria consumed only 16% of glucose while 45% was exported as lactate by aerobic glycolysis. The remaining 39% of glucose was shunted to pentose phosphates presumably for de novo nucleotide synthesis, but the proportion metabolized through the oxidative PPP vs. the reverse non-oxidative PPP could not be determined. The lactate exported as M1 (2.5%) and M3 (1.2%) was attributed to malic enzyme, which was responsible for 7.8% of pyruvate production (vs. 92.2% by glycolysis). The updated model is more broadly applicable to different cell types by considering bi-directional flux through the non-oxidative PPP. Its application to cultured neurons utilizing glucose as the sole exogenous substrate has demonstrated substantial oxygen-independent glucose utilization by aerobic glycolysis as well as the oxidative PPP and/or reverse non-oxidative PPP, but negligible glucose consumption by the pentose cycle.
Asunto(s)
Isótopos de Carbono/metabolismo , Cerebelo/citología , Cerebelo/metabolismo , Metabolismo Energético/fisiología , Análisis de Flujos Metabólicos/métodos , Neuronas/metabolismo , Animales , Animales Recién Nacidos , Isótopos de Carbono/análisis , Células Cultivadas , RatasRESUMEN
Glycolysis, mitochondrial substrate oxidation, and the pentose phosphate pathway (PPP) are critical for neuronal bioenergetics and oxidation-reduction homeostasis, but quantitating their fluxes remains challenging, especially when processes such as hexose phosphate (i.e., glucose/fructose-6-phosphate) recycling in the PPP are considered. A hexose phosphate recycling model was developed which exploited the rates of glucose consumption, lactate production, and mitochondrial respiration to infer fluxes through the major glucose consuming pathways of adherent cerebellar granule neurons by replicating [(13)C]lactate labeling from metabolism of [1,2-(13)C2]glucose. Flux calculations were predicated on a steady-state system with reactions having known stoichiometries and carbon atom transitions. Non-oxidative PPP activity and consequent hexose phosphate recycling, as well as pyruvate production by cytoplasmic malic enzyme, were optimized by the model and found to account for 28 ± 2% and 7.7 ± 0.2% of hexose phosphate and pyruvate labeling, respectively. From the resulting fluxes, 52 ± 6% of glucose was metabolized by glycolysis, compared to 19 ± 2% by the combined oxidative/non-oxidative pentose cycle that allows for hexose phosphate recycling, and 29 ± 8% by the combined oxidative PPP/de novo nucleotide synthesis reactions. By extension, 62 ± 6% of glucose was converted to pyruvate, the metabolism of which resulted in 16 ± 1% of glucose oxidized by mitochondria and 46 ± 6% exported as lactate. The results indicate a surprisingly high proportion of glucose utilized by the pentose cycle and the reactions synthesizing nucleotides, and exported as lactate. While the in vitro conditions to which the neurons were exposed (high glucose, no lactate or other exogenous substrates) limit extrapolating these results to the in vivo state, the approach provides a means of assessing a number of metabolic fluxes within the context of hexose phosphate recycling in the PPP from a minimal set of measurements.
Asunto(s)
Isótopos de Carbono/metabolismo , Hexosas/metabolismo , Modelos Biológicos , Neuronas/metabolismo , Vía de Pentosa Fosfato , Animales , Cerebelo/citología , Cerebelo/metabolismo , Cromatografía Liquida , Gránulos Citoplasmáticos/metabolismo , Método de Montecarlo , Ratas , Ratas Wistar , Espectrometría de Masas en TándemRESUMEN
Hypoxia is a common feature of solid tumors, and the extent of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective molecular target for anticancer drug discovery directed at tumor hypoxia. A natural product chemistry-based approach was employed to discover small molecule inhibitors of HIF-1. Bioassay-guided isolation of an active lipid extract of the tropical legumaceous plant Lonchocarpus glabrescens and structure elucidation afforded two new HIF-1 inhibitors: alpinumisoflavone (compound 1) and 4'-O-methylalpinumisoflavone (compound 2). In human breast tumor T47D cells, compounds 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC(50) values of 5 and 0.6 mum, respectively. At the concentrations that in hibited HIF-1 activation, compound 2 inhibited hypoxic induction of HIF-1 target genes (CDKN1A, GLUT-1, and VEGF), tumor angiogenesis in vitro, cell migration, and chemotaxis. Compound 2 inhibits HIF-1 activation by blocking the induction of nuclear HIF-1alpha protein, the oxygen-regulated subunit that controls HIF-1 activity. Mechanistic studies indicate that, unlike rotenone and other mitochondrial inhibitors, compound 2 represents the first small molecule that inhibits HIF-1 activation by simultaneously suppressing mitochondrial respiration and disrupting protein translation in vitro. This unique mechanism distinguishes compound 2 from other small molecule HIF-1 inhibitors that are simple mitochondrial inhibitors or flavanoid-based protein kinase inhibitors.