Targeted therapies of melanoma are of urgent need considering the resistance of this aggressive type of cancer to chemotherapeutics. The voltage-dependent anion channel 1 (VDAC1)-hexokinase-II (HK-II) complex is an emerging target for novel anticancer therapies based on induced mitochondria-mediated apoptosis. The low cell membrane permeability of the anticancer 12-mer peptide N-Ter (RDVFTKGYGFGL) derived from the N-terminal fragment of the VDAC1 protein impedes the intracellular targeting. Here, novel multiblock VDAC1-derived cationic amphiphilic peptides (referred to as Pal-N-Ter-TAT, pFL-N-Ter-TAT, and Pal-pFL-N-Ter-TAT) are designed with a self-assembly propensity and cell-penetrating properties. The created multiblock amphiphilic peptides of partial α-helical conformations form nanoparticles of ellipsoid-like shapes and are characterized by enhanced cellular uptake. The amphiphilic peptides can target mitochondria and dissociate the VDAC1-HK-II complex at the outer mitochondrial membrane, which result in mitochondria-mediated apoptosis. The latter is associated with decrease of the mitochondrial membrane potential, cytochrome c release, and changes of the expression levels of the apoptotic proteins in A375 melanoma cells. Importantly, the mitochondrial VDAC1-derived amphiphilic peptides have a comparable IC50 value for melanoma cells to a small-molecule drug, sorafenib, which has been previously used in clinical trials for melanoma. These results demonstrate the potential of the designed peptide constructs for efficient melanoma inhibition.

• MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• hexokinasa metabolismus   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální membrány účinky léků   MeSH
• mitochondrie účinky léků   MeSH
• nádorové buněčné linie MeSH
• napětím ovládaný aniontový kanál 1 metabolismus   MeSH
• peptidy farmakologie   MeSH
• povrchově aktivní látky farmakologie   MeSH
• sekvence aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The identification of compounds which protect the double-membrane of mitochondrial organelles from disruption by toxic confomers of amyloid proteins may offer a therapeutic strategy to combat human neurodegenerative diseases. Here, we exploited an extract from the marine brown seaweed Padina pavonica (PPE) as a vital source of natural bioactive compounds to protect mitochondrial membranes against insult by oligomeric aggregates of the amyloidogenic proteins amyloid-β (Aβ), α-synuclein (α-syn) and tau, which are currently considered to be major targets for drug discovery in Alzheimer's disease (AD) and Parkinson's disease (PD). We show that PPE manifested a significant inhibitory effect against swelling of isolated mitochondria exposed to the amyloid oligomers, and attenuated the release of cytochrome c from the mitochondria. Using cardiolipin-enriched synthetic lipid membranes, we also show that dye leakage from fluorophore-loaded vesicles and formation of channel-like pores in planar bilayer membranes are largely prevented by incubating the oligomeric aggregates with PPE. Lastly, we demonstrate that PPE curtails the ability of Aβ42 and α-syn monomers to self-assemble into larger β-aggregate structures, as well as potently disrupts their respective amyloid fibrils. In conclusion, the mito-protective and anti-aggregator biological activities of Padina pavonica extract may be of therapeutic value in neurodegenerative proteinopathies, such as AD and PD.

• MeSH
• alfa-synuklein metabolismus   toxicita   MeSH
• amyloidní beta-protein metabolismus   toxicita   MeSH
• lidé MeSH
• lipidové dvojvrstvy chemie   MeSH
• mitochondriální membrány účinky léků   patologie   MeSH
• mořské řasy chemie   MeSH
• neuroprotektivní látky chemie   farmakologie   MeSH
• peptidové fragmenty metabolismus   toxicita   MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• Phaeophyceae chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Hypoxia causes mitochondrial cristae widening, enabled by the ~20% degradation of Mic60/mitofilin, with concomitant clustering of the MICOS complex, reflecting the widening of crista junctions (outlets) (Plecitá-Hlavatá et al. FASEB J., 2016 30:1941-1957). Attempting to accelerate metabolism by the addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to HepG2 cells pre-adapted to hypoxia, we found cristae narrowing by transmission electron microscopy. Glycolytic HepG2 cells, which downregulate hypoxic respiration, instantly increased respiration with dm2OG. Changes in intracristal space (ICS) morphology were also revealed by 3D super-resolution microscopy using Eos-conjugated ICS-located lactamase-β. Cristae topology was resolved in detail by focused-ion beam/scanning electron microscopy (FIB/SEM). The spatial relocations of key cristae-shaping proteins were indicated by immunocytochemical stochastic 3D super-resolution microscopy (dSTORM), while analyzing inter-antibody-distance histograms: i) ATP-synthase dimers exhibited a higher fraction of shorter inter-distances between bound F1-α primary Alexa-Fluor-647-conjugated antibodies, indicating cristae narrowing. ii) Mic60/mitofilin clusters (established upon hypoxia) decayed, restoring isotropic random Mic60/mitofilin distribution (a signature of normoxia). iii) outer membrane SAMM50 formed more focused clusters. Less abundant fractions of higher ATP-synthase oligomers of hypoxic samples on blue-native electrophoresis became more abundant fractions at the high dm2OG load and at normoxia. This indicates more labile ATP-synthase dimeric rows established at crista rims upon hypoxia, strengthened at normoxia or dm2OG-substrate load. Hypothetically, the increased Krebs substrate load stimulates the cross-linking/strengthening of rows of ATP-synthase dimers at the crista rims, making them sharper. Crista narrowing ensures a more efficient coupling of proton pumping to ATP synthesis. We demonstrated that cristae morphology changes even within minutes.

• MeSH
• buněčné dýchání MeSH
• buňky Hep G2 MeSH
• dimerizace MeSH
• hypoxie MeSH
• kyseliny ketoglutarové farmakologie   MeSH
• lidé MeSH
• mitochondriální membrány účinky léků   ultrastruktura   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondriální protonové ATPasy metabolismus   MeSH
• mitochondrie ultrastruktura   MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pancreatic β-cells are vulnerable to oxidative stress due to their low content of redox buffers, such as glutathione, but possess a rich content of thioredoxin, peroxiredoxin, and other proteins capable of redox relay, transferring redox signaling. Consequently, it may be predicted that cytosolic antioxidants could interfere with the cytosolic redox signaling and should not be recommended for any potential therapy. In contrast, mitochondrial matrix-targeted antioxidants could prevent the primary oxidative stress arising from the primary superoxide sources within the mitochondrial matrix, such as at the flavin (IF) and ubiquinone (IQ) sites of superoxide formation within respiratory chain complex I and the outer ubiquinone site (IIIQ) of complex III. Therefore, using time-resolved confocal fluorescence monitoring with MitoSOX Red, we investigated various effects of mitochondria-targeted antioxidants in model pancreatic β-cells (insulinoma INS-1E cells) and pancreatic islets. Both SkQ1 (a mitochondria-targeted plastoquinone) and a suppressor of complex III site Q electron leak (S3QEL) prevented superoxide production released to the mitochondrial matrix in INS-1E cells with stimulatory glucose, where SkQ1 also exhibited an antioxidant role for UCP2-silenced cells. SkQ1 acted similarly at nonstimulatory glucose but not in UCP2-silenced cells. Thus, UCP2 can facilitate the antioxidant mechanism based on SkQ1+ fatty acid anion- pairing. The elevated superoxide formation induced by antimycin A was largely prevented by S3QEL, and that induced by rotenone was decreased by SkQ1 and S3QEL and slightly by S1QEL, acting at complex I site Q. Similar results were obtained with the MitoB probe, for the LC-MS-based assessment of the 4 hr accumulation of reactive oxygen species within the mitochondrial matrix but for isolated pancreatic islets. For 2 hr INS-1E incubations, some samples were influenced by the cell death during the experiment. Due to the frequent dependency of antioxidant effects on metabolic modes, we suggest a potential use of mitochondria-targeted antioxidants for the treatment of prediabetic states after cautious nutrition-controlled tests. Their targeted delivery might eventually attenuate the vicious spiral leading to type 2 diabetes.

• MeSH
• antioxidancia farmakologie   MeSH
• beta-buňky účinky léků   metabolismus   patologie   MeSH
• fenantridiny MeSH
• kultivované buňky MeSH
• mitochondriální membrány účinky léků   metabolismus   patologie   MeSH
• mitochondrie účinky léků   metabolismus   patologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• organofosforové sloučeniny MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• uncoupling protein 2 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

CONTEXT: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP in vitro. OBJECTIVES: The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures. MATERIALS AND METHODS: Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24 h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1). RESULTS: APAP from concentrations of 2.5 and 0.75 mmol/L induced a decrease in viability of rat (p < 0.001) and mouse (p < 0.001) hepatocytes (WST-1), respectively. In contrast to rat hepatocytes, there was no activation of caspase-3 in mouse hepatocytes after APAP treatment. Earlier damage to plasma membrane and faster depletion of reduced glutathione were detected in mouse hepatocytes. Mouse hepatocytes showed increased glutamate + malate-driven respiration in state 4 and higher susceptibility of the outer mitochondrial membrane (OMM) to APAP-induced injury. CONCLUSION: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.

• MeSH
• biologické markery metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• druhová specificita MeSH
• glutathion metabolismus   MeSH
• hepatocyty cytologie   účinky léků   metabolismus   MeSH
• jaterní mitochondrie účinky léků   enzymologie   metabolismus   MeSH
• kultivované buňky MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální membrány účinky léků   metabolismus   MeSH
• myši inbrední C57BL MeSH
• neopioidní analgetika škodlivé účinky   MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• paracetamol škodlivé účinky   MeSH
• peroxidace lipidů účinky léků   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku agonisté   metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Pharmacological preconditioning by diazoxide and a model of experimental streptozotocin-induced acute diabetes mellitus (STZ-DM) provided similar levels of cardioprotection assessed as limiting myocardial infarct size. The aim was to explore the possibility of existence of another in vitro mechanism, which could be contributory to cardioprotection mediated by diazoxide treatment. Mitochondrial membrane fluidity and ATP synthase activity in isolated heart mitochondria were determined under the influence of two factors, STZ-DM condition and treatment with diazoxide. Both factors independently increased the ATP synthase activity (p<0.05), as no interaction effect was observed upon the combination of STZ-DM with diazoxide. On the other hand, the mitochondrial membrane fluidity was significantly increased by STZ-DM only; no significant main effect for diazoxide was found. Based on the results from measurements of enzyme kinetics, we assume a direct interaction of diazoxide with the molecule of ATP synthase stimulated its activity by noncompetitive activation. Our present work revealed, for the first time, that cardioprotection induced by diazoxide may not be caused exclusively by mitochondrial K(ATP) opening, but presumably also by a direct interaction of diazoxide with ATP synthase, although the mechanisms for achieving this activation cannot be fully delineated.

• MeSH
• diazoxid farmakologie   terapeutické užití   MeSH
• experimentální diabetes mellitus enzymologie   MeSH
• fluidita membrány účinky léků   MeSH
• mitochondriální membrány účinky léků   MeSH
• mitochondriální protonové ATPasy metabolismus   MeSH
• nemoci srdce prevence a kontrola   MeSH
• potkani Wistar MeSH
• sukcinátdehydrogenasa antagonisté a inhibitory   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• antioxidancia metabolismus   farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buněčné dýchání účinky léků   MeSH
• buněčné linie MeSH
• energetický metabolismus účinky léků   MeSH
• glutathion metabolismus   MeSH
• homeostáza MeSH
• kinetika MeSH
• lidé MeSH
• mitochondriální membrány účinky léků   metabolismus   MeSH
• molekulární struktura MeSH
• molekulová hmotnost MeSH
• oxidace-redukce MeSH
• oxidační stres účinky léků   MeSH
• polyethylenimin chemie   toxicita   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• spotřeba kyslíku účinky léků   MeSH
• transfekce metody   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Digitonin solubilizes mitochondrial membrane, breaks the integrity of the respiratory chain and releases two mobile redox-active components: coenzyme Q (CoQ) and cytochrome c (cyt c). In the present study we report the inhibition of glycerol-3-phosphate- and succinate-dependent oxygen consumption rates by digitonin treatment. Our results show that the inhibition of oxygen consumption rates is recovered by the addition of exogenous synthetic analog of CoQ idebenone (hydroxydecyl-ubiquinone; IDB) and cyt c. Glycerol-3-phosphate oxidation rate is recovered to 148 % of control values, whereas succinate-dependent oxidation rate only to 68 %. We find a similar effect on the activities of glycerol-3-phosphate and succinate cytochrome c oxidoreductase. Our results also indicate that succinate-dependent oxidation is less sensitive to digitonin treatment and less activated by IDB in comparison with glycerol-3-phosphate-dependent oxidation. These findings might indicate the different mechanism of the electron transfer from two flavoprotein-dependent dehydrogenases (glycerol-3-phosphate dehydrogenase and succinate dehydrogenase) localized on the outer and inner face of the inner mitochondrial membrane, respectively.

• MeSH
• cytochromy c metabolismus   MeSH
• digitonin farmakologie   MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• glycerolfosfáty metabolismus   MeSH
• hypertyreóza metabolismus   MeSH
• jaterní mitochondrie metabolismus   účinky léků   MeSH
• kinetika MeSH
• krysa rodu rattus MeSH
• kyselina jantarová metabolismus   MeSH
• mitochondriální membrány metabolismus   účinky léků   MeSH
• modely nemocí na zvířatech MeSH
• obnova funkce MeSH
• oxidace-redukce MeSH
• potkani Wistar MeSH
• spotřeba kyslíku účinky léků   MeSH
• sukcinátcytochrom c oxidoreduktasa metabolismus   MeSH
• ubichinon analogy a deriváty   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of the work was early identification of preventable risk factors connected with the consumers usage of products of everyday use, such as cosmetics, toys and children products, and other materials intended for contact with human skin. The risk factor is represented by substances with irritation potential and subsequent possible sensitisation, resulting in negative impact on human physical and psychical health with social and societal consequences. The legislation for cosmetics, chemical substances and other products requires for hazard identification the application of alternative toxicological methods in vitro without the use of animals. For this reason we used a battery of alternative assays in vitro, based on cell cultures. Progressive methods of molecular biology, based on fluorimetry and fluorescence, were employed for identification of early morphological and functional changes on cellular level. Four colorants frequently used in cosmetics (P-WS Caramel, Chlorophyllin, Unicert Red K 7054-J and Unicert Red K 7008-J) were tested on cell line NIH3T3 (mouse fibroblast cell) and 3T3 Balb/c with/without UV irradiation (dose 5 J cm(-2)). Fluorescence methods for the study of cell damage using fluorescence probes offer results for the evaluation of cytotoxicity and cell viability of adherent cells. We detected intracellular production of ROS investigated by molecular probe CM-H(2)DCFDA, which is primarily sensitive to the increased production of hydrogen peroxide or its downstream products. Toxic effects on the cellular level were identified by viability tests using Neutral Red uptake and MTT assay, where the live cells reduce yellow soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals. The reaction was investigated on mitochondrial membrane of living cells and the type of cell death was determined using Apoptosis detection kit. Cytotoxicity tests revealed health risks of using Chlorophyllin and Unicert Red K 7054-J.

• MeSH
• barvicí látky toxicita   MeSH
• buněčná smrt účinky léků   MeSH
• buňky BALB 3T3 MeSH
• buňky NIH 3T3 MeSH
• fluorescence MeSH
• fluorometrie metody   MeSH
• fototoxická dermatitida etiologie   MeSH
• kosmetické přípravky chemie   toxicita   MeSH
• lidé MeSH
• mitochondriální membrány účinky léků   metabolismus   MeSH
• myši MeSH
• peroxid vodíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• testy toxicity metody   MeSH
• ultrafialové záření MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Photodynamic therapy (PDT) has been approved as proper and effective kind of treatment for certain types of cancer and non-malignant diseases. We tested photodynamic effects on G361 human melanoma cells sensitized by zinc-5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrine (ZnTPPS(4)), chloraluminium phtalocyanine disulfonate (ClAlPcS(2)) and 5-aminolevulinic acid (ALA). In particular, we examined the PDT efficiency depending on applied light dose (0.8; 1.7; 3.3; 6.6; 13.2; 26.4Jcm(-2)). The DNA gel electrophoresis, methylthiazol tetrazolium bromide (MTT) viability test, fluorescent microscopy using calcein AM and propidium iodide (PI) staining, and rhodamine 123 mitochondrial membrane potential assay were performed to detect and evaluate the cell death process. We also measured the time course of reactive oxygen species (ROS) production and its dependence on sensitizer concentration within continuously irradiated sensitized cells. In conclusion, these results demonstrate most significant phototoxic effect of ClAlPcS(2)-PDT in spite of significantly higher ROS production induced by ZnTPPS(4)-PDT on G361 cells. On the other hand, ALA-PDT has a minimal photoeffect and induces negligible ROS formation in G361 cells at the conditions described below.

• MeSH
• barvicí látky MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fluorescenční barviva MeSH
• fluorescenční mikroskopie MeSH
• fotochemoterapie MeSH
• fotosenzibilizující látky farmakologie   MeSH
• fragmentace DNA účinky léků   účinky záření   MeSH
• indoly farmakologie   MeSH
• kyselina aminolevulová farmakologie   MeSH
• lidé MeSH
• melanom experimentální terapie   MeSH
• membránové potenciály účinky léků   MeSH
• metaloporfyriny farmakologie   MeSH
• mitochondriální membrány účinky léků   MeSH
• nádorové buněčné linie MeSH
• organokovové sloučeniny farmakologie   MeSH
• propidium MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• rhodamin 123 MeSH
• světlo MeSH
• tetrazoliové soli MeSH
• thiazoly MeSH
• vztah dávky záření a odpovědi MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH