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1.
Cancer Metab ; 11(1): 4, 2023 Feb 20.
Article in English | MEDLINE | ID: mdl-36805760

ABSTRACT

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.

2.
Org Lett ; 24(40): 7446-7449, 2022 10 14.
Article in English | MEDLINE | ID: mdl-36194640

ABSTRACT

We report the formation of zinc reagents by the reaction of styrylsulfonium salts with zinc powder. Transition metals and other additives are not required for promoting zincation. Zincation tolerates a variety of sensitive functional groups, including esters, bromides, and boronic esters, and proceeds with complete retention of stereochemistry. This method presents a practical approach to the formation of zinc reagents that can be used in a variety of functionalizations, such as halogenation, carboxylation, and Negishi cross-couplings.

3.
Comput Biol Med ; 100: 305-315, 2018 09 01.
Article in English | MEDLINE | ID: mdl-29397919

ABSTRACT

Electrogastrography (EGG) is a noninvasive technique for recording the myoelectrical activity of the stomach. An electrogastrographic signal recorded by using a four-channel system with electrodes placed on the surface of the skin is a mixture of a low-frequency gastric pacesetter potential known as a slow wave, electrical activity from other organs, and random noise. The aim of this work was to investigate the possibility of detecting the propagation of the gastric slow wave from multichannel EGG data. Noise-assisted multivariate empirical mode decomposition (NA-MEMD) and cross-covariance analysis (CCA) are proposed as new detection tools. NA-MEMD was applied to attenuate the noise and extract the EGG signal from four channels, while CCA was performed to assess the time shift between the EGG signal channels. Validation of the method was performed using synthetic EGG signals and the methodology was tested on four young, healthy adults. After validation, the proposed method was applied for two kinds of human EGG data: 10-min (short) EGG data from the preprandial phase and 90-120-min (long) EGG data from the preprandial phase as well as the postprandial phase. The results obtained for both synthetic and human EGG data confirm that the proposed method could be a useful tool for assessing the propagation of slow waves. The time shift calculation from the preprandial phase of the EGG examination yielded more consistent results than the postprandial phase. The mean value of the slow wave time lag between neighbouring channels for synthetic data was found to be 4.99±0.47 s. In addition, it was confirmed that the proposed method, that is, NA-MEMD and CCA together, are robust to noise.


Subject(s)
Electromyography/methods , Signal Processing, Computer-Assisted , Stomach/physiology , Adult , Female , Humans , Male
4.
Cell Death Differ ; 25(3): 542-572, 2018 03.
Article in English | MEDLINE | ID: mdl-29229998

ABSTRACT

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.


Subject(s)
Mitochondria/metabolism , Mitochondria/pathology , Models, Biological , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Animals , Humans
5.
Neurochem Int ; 109: 54-67, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28412312

ABSTRACT

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.


Subject(s)
Carbon Isotopes/metabolism , Cerebellum/cytology , Cerebellum/metabolism , Energy Metabolism/physiology , Metabolic Flux Analysis/methods , Neurons/metabolism , Animals , Animals, Newborn , Carbon Isotopes/analysis , Cells, Cultured , Rats
6.
Neurochem Int ; 93: 26-39, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26723542

ABSTRACT

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.


Subject(s)
Carbon Isotopes/metabolism , Hexoses/metabolism , Models, Biological , Neurons/metabolism , Pentose Phosphate Pathway , Animals , Cerebellum/cytology , Cerebellum/metabolism , Chromatography, Liquid , Cytoplasmic Granules/metabolism , Monte Carlo Method , Rats , Rats, Wistar , Tandem Mass Spectrometry
7.
J Nat Prod ; 78(12): 3018-23, 2015 Dec 24.
Article in English | MEDLINE | ID: mdl-26637046

ABSTRACT

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.


Subject(s)
Alkaloids/pharmacology , Anti-Infective Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Quinones/pharmacology , Animals , Benzoquinones , Cell Cycle/drug effects , Cell Division , Cell Respiration/drug effects , Electron Transport , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Mitochondria/metabolism , Molecular Structure , Naphthyridines , Oxidation-Reduction , Oxygen , Rats , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae , Superoxides/metabolism
8.
Mar Drugs ; 13(3): 1552-68, 2015 Mar 20.
Article in English | MEDLINE | ID: mdl-25803180

ABSTRACT

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.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Breast Neoplasms/drug therapy , Lipids/pharmacology , Neovascularization, Pathologic/drug therapy , Thiazoles/pharmacology , Angiogenesis Inhibitors/administration & dosage , Breast Neoplasms/pathology , Cell Hypoxia/drug effects , Cell Line, Tumor , Electron Transport Complex I/drug effects , Electron Transport Complex I/metabolism , Female , Humans , Hypoxia-Inducible Factor 1/metabolism , Inhibitory Concentration 50 , Lipids/administration & dosage , Mitochondria/drug effects , Mitochondria/metabolism , Signal Transduction/drug effects , Thiazoles/administration & dosage , Vascular Endothelial Growth Factor A/metabolism
9.
J Nat Prod ; 77(1): 111-7, 2014 Jan 24.
Article in English | MEDLINE | ID: mdl-24328138

ABSTRACT

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.


Subject(s)
Caulophyllum/chemistry , Cell Respiration/drug effects , Dietary Supplements/toxicity , Membrane Potential, Mitochondrial/drug effects , Oleanolic Acid/analogs & derivatives , Saponins/toxicity , Dose-Response Relationship, Drug , Humans , Molecular Structure , Oleanolic Acid/chemistry , Oleanolic Acid/toxicity , Phytotherapy , Saponins/chemistry , United States
10.
Bioorg Med Chem ; 21(7): 1795-803, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23434131

ABSTRACT

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.


Subject(s)
Aegle/chemistry , Coumarins/chemistry , Coumarins/toxicity , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Mitochondria/drug effects , Plant Extracts/chemistry , Plant Extracts/toxicity , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/toxicity , Breast/drug effects , Breast/pathology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Respiration/drug effects , Cell Survival/drug effects , Coumarins/isolation & purification , Coumarins/pharmacology , Dietary Supplements/adverse effects , Dietary Supplements/analysis , Female , Humans , Hypoxia-Inducible Factor 1/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Plant Extracts/isolation & purification , Plant Extracts/pharmacology
11.
J Nat Prod ; 75(12): 2216-22, 2012 Dec 28.
Article in English | MEDLINE | ID: mdl-23245650

ABSTRACT

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.


Subject(s)
Clusiaceae/chemistry , Glycolysis/drug effects , Phloroglucinol/analogs & derivatives , Phloroglucinol/isolation & purification , Phloroglucinol/pharmacology , Cell Survival/drug effects , Female , Glucose/metabolism , Humans , Molecular Structure , Neoplasms/metabolism , Phloroglucinol/chemistry , Rotenone/pharmacology
12.
PLoS One ; 7(11): e48912, 2012.
Article in English | MEDLINE | ID: mdl-23139825

ABSTRACT

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.


Subject(s)
Arrestin/metabolism , G-Protein-Coupled Receptor Kinases/metabolism , Neostriatum/cytology , Neostriatum/enzymology , Animals , Cell Nucleus/enzymology , Cholinergic Neurons/cytology , Cholinergic Neurons/enzymology , Female , Humans , Interneurons/cytology , Interneurons/enzymology , Isoenzymes/metabolism , Male , Middle Aged , Phosphorylation , Protein Transport , Rats , Rats, Sprague-Dawley , Subcellular Fractions/enzymology
13.
J Nat Prod ; 75(9): 1553-9, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-22938093

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Porifera/chemistry , Quinones/isolation & purification , Quinones/pharmacology , Animals , Antineoplastic Agents/chemistry , Drug Screening Assays, Antitumor , Female , Humans , Inhibitory Concentration 50 , Iron Chelating Agents/pharmacology , Marine Biology , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Quinones/chemistry
14.
J Neurochem ; 119(5): 1137-50, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21951169

ABSTRACT

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.


Subject(s)
Cerebellar Cortex/chemistry , Cytoplasmic Granules/chemistry , Digitonin/pharmacology , Neurons/chemistry , Voltage-Dependent Anion Channel 1/chemistry , bcl-2-Associated X Protein/chemistry , Animals , Animals, Newborn , Cerebellar Cortex/cytology , Cerebellar Cortex/metabolism , Cholic Acids/pharmacology , Cytoplasmic Granules/metabolism , Neurons/classification , Neurons/metabolism , Primary Cell Culture , Rats , Rats, Wistar , Solubility , Voltage-Dependent Anion Channel 1/physiology , bcl-2-Associated X Protein/physiology
15.
J Nat Prod ; 74(2): 240-8, 2011 Feb 25.
Article in English | MEDLINE | ID: mdl-21214226

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Phytogenic , Cell Respiration/drug effects , Coumarins , Hypoxia-Inducible Factor 1/drug effects , Mammea/chemistry , Algorithms , Antineoplastic Agents, Phytogenic/chemical synthesis , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Coumarins/chemical synthesis , Coumarins/chemistry , Coumarins/isolation & purification , Coumarins/pharmacology , Dominica , Drug Screening Assays, Antitumor , Humans , Male , Molecular Structure , Plant Bark/chemistry , Prenylation , Structure-Activity Relationship
16.
J Nat Prod ; 73(11): 1868-72, 2010 Nov 29.
Article in English | MEDLINE | ID: mdl-20929261

ABSTRACT

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.


Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Coumarins/isolation & purification , Coumarins/pharmacology , Hypoxia-Inducible Factor 1/drug effects , Mammea/chemistry , Mitochondria/drug effects , Plant Bark , Animals , Antineoplastic Agents, Phytogenic/chemistry , Coumarins/chemistry , Disease Models, Animal , Dominica , Electron Transport , Female , Humans , Mitochondria/metabolism , Molecular Structure , Plant Bark/chemistry , Prenylation , Vascular Endothelial Growth Factors/drug effects
17.
Bioorg Med Chem ; 18(16): 5988-94, 2010 Aug 15.
Article in English | MEDLINE | ID: mdl-20637638

ABSTRACT

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.


Subject(s)
Electron Transport Complex I/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Hypoxia-Inducible Factor 1/antagonists & inhibitors , Porifera/chemistry , Thiazoles/pharmacology , Animals , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Electron Transport Complex I/metabolism , Enzyme Inhibitors/isolation & purification , Female , Gene Expression Regulation/drug effects , Humans , Hypoxia-Inducible Factor 1/genetics , Hypoxia-Inducible Factor 1/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neovascularization, Pathologic/drug therapy , Neurons/drug effects , Rats , Thiazoles/isolation & purification
18.
J Nat Prod ; 73(5): 956-61, 2010 May 28.
Article in English | MEDLINE | ID: mdl-20423107

ABSTRACT

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.


Subject(s)
Acetogenins/isolation & purification , Acetogenins/pharmacology , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Asimina/chemistry , Glucose Transporter Type 1/drug effects , Hypoxia-Inducible Factor 1/drug effects , Neovascularization, Physiologic/drug effects , Plants, Medicinal/chemistry , Vascular Endothelial Growth Factors/drug effects , Acetogenins/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Complementary Therapies , Drug Screening Assays, Antitumor , Glucose Transporter Type 1/analysis , Glucose Transporter Type 1/genetics , Humans , Inhibitory Concentration 50 , Molecular Structure , Vascular Endothelial Growth Factors/analysis , Vascular Endothelial Growth Factors/genetics
19.
J Nat Prod ; 72(12): 2104-9, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19921787

ABSTRACT

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.


Subject(s)
Caulerpa/chemistry , Hypoxia-Inducible Factor 1/drug effects , Indoles/pharmacology , Coloring Agents/chemistry , Coloring Agents/pharmacology , Electron Transport Complex I/antagonists & inhibitors , Female , Humans , Indoles/chemistry , Indoles/isolation & purification , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Vascular Endothelial Growth Factor A/metabolism
20.
J Nat Prod ; 72(11): 1927-36, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19845338

ABSTRACT

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.


Subject(s)
Hypoxia-Inducible Factor 1/antagonists & inhibitors , Hypoxia-Inducible Factor 1/metabolism , Mitochondria/metabolism , Porifera/chemistry , Pyrroles/isolation & purification , Pyrroles/pharmacology , Animals , Cell Respiration/drug effects , Female , Humans , Marine Biology , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Palau , Pyrroles/chemistry
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