cytochrome-c-t and nitroxyl

Combined superoxide dismutase (SOD)/catalase mimetics have attracted much attention because of their efficacy against reactive oxygen species-associated diseases; however, their application is often limited owing to their poor stability and the absence of favorable grafting sites. To address this, we developed a new class of SOD/catalase mimetics based on hybrid nanoflowers, which exhibit superior stability and possess the desired grafting sites for drugs and endogenous molecules.. The H. Herein, a comparison of Michaelis constants showed that the hybrid nanoflower, a catalase mimetics, outperforms the native catalase. Acting as a "better-than-nature" enzyme mimetics, the hybrid nanoflower with superior stability and desired ligand grafting sites will find widespread utilization in the medical sciences.

Topics: Catalase; Copper; Cytochromes c; Electron Spin Resonance Spectroscopy; Hydrogen Peroxide; Kinetics; Microscopy, Electron, Scanning; Molecular Mimicry; Nanostructures; Nitrogen Oxides; Oxygen; Reproducibility of Results; Serum Albumin, Human; Spectroscopy, Fourier Transform Infrared; Superoxide Dismutase; Xanthine Oxidase

A synthetic amphiphilic block copolymer, Pluronic, is a potent chemosensitizer of multidrug resistant (MDR) cancers that has shown promise in clinical trials. It has unique activities in MDR cells, which include a decrease in ATP pools and inhibition of P-glycoprotein (Pgp) resulting in increased drug accumulation in cells. This work demonstrates that Pluronic rapidly (15min) translocates into MDR cells and co-localizes with the mitochondria. It inhibits complex I and complex IV of the mitochondria respiratory chain, decreases oxygen consumption and causes ATP depletion in MDR cells. These effects are selective and pronounced for MDR cells compared to non-MDR counterparts and demonstrated for both drug-selected and Pgp-transfected cell models. Furthermore, inhibition of Pgp functional activity also abolishes the effects of Pluronic on intracellular ATP levels in MDR cells suggesting that Pgp contributes to increased responsiveness of molecular "targets" of Pluronic in the mitochondria of MDR cells. The Pluronic-caused impairment of respiration in mitochondria of MDR cells is accompanied with a decrease in mitochondria membrane potential, production of ROS, and release of cytochrome c. Altogether these effects eventually enhance drug-induced apoptosis and contribute to potent chemosensitization of MDR tumors by Pluronic.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cytochromes c; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Mitochondria; Nitrogen Oxides; Oxygen Consumption; Phosphorylation; Poloxamer; Reactive Oxygen Species

We have shown that heat stress or a superoxide dismutase mimic nitroxide, Tempo, induces apoptosis, while their combination causes nonapoptotic cell death; however, the underlying mechanism for this switch remains unclear. Here we identified for the first time that 10 mM Tempo present during heating at 44°C for 30 min rapidly induced autophagy in U937 leukemic cells in spite of Bax activation and mitochondrial outer membrane (MOM) permeabilization. This co-treatment inhibited the processing of heat-activated procaspases-2, -8, -9 and -3 into active small subunits, leading to the inhibition of caspase-dependent apoptosis, and instead caused the induction of autophagy. The inactivation of caspases, a key event, could result from oxidation of active-site-CysSH of all caspases by a prooxidant oxo-ammonium cation, an intermediate derived Tempo during dismutation of heat-induced superoxide anion. In addition, the co-treatment caused mitochondrial calcium overloads, the mitochondrial inner membrane permeabilization, profound mitochondrial dysfunction, and liberation of Beclin 1 from the Bcl-2/Beclin 1 complex, all of which contributed to induction of autophagy. These autophagic cells underwent propidium iodide-positive necrosis in a delayed fashion, leading to the complete proliferative inhibition. Remarkably, ruthenium red and BAPTA, which interfere with mitochondrial calcium uptake, facilitated autophagic necrotic death. Cyclosporin A, which binds to cyclophilin D, had a similar necrotic effect. 3-Methyladenine facilitated the necrosis of autophagic cells. In contrast, 5 mM Tempo-44°C/10 min or 44°C/30 min induced Bax-mediated MOM permeabilization and caspase-dependent apoptosis more potently than Tempo alone. Thus, Tempo is a unique thermosensitizer to synergistically induce apoptosis and autophagic cell death.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Caspase 8; Caspase 9; Caspases; Cyclic N-Oxides; Cytochromes c; Fluorescein-5-isothiocyanate; Heat-Shock Response; Hot Temperature; Humans; Immunoblotting; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Mitochondrial Membranes; Nitrogen Oxides; Signal Transduction; Superoxide Dismutase; U937 Cells

We report methodology which combines recently developed dielectric resonator-based, rapid-mix, stopped-flow EPR (appropriate for small, aqueous, lossy samples) with rapid scanning of the external (Zeeman) magnetic field where the scanning is preprogrammed to occur at selected times after the start of flow. This methodology gave spectroscopic information complementary to that obtained by stopped-flow EPR at single fields, and with low reactant usage, it yielded more graphic insight into the time evolution of radical and spin-labeled species. We first used the ascorbyl radical as a test system where rapid scans triggered after flow was stopped provided "snapshots" of simultaneously evolving and interacting radical species. We monitored ascorbyl radical populations either as brought on by biologically damaging peroxynitrite oxidant or as chemically and kinetically interacting with a spectroscopically overlapping nitroxide radical. In a different biophysical application, where a spin-label lineshape reflected rapidly changing molecular dynamics of folding spin-labeled protein, rapid scan spectra were taken during flow with different flow rates and correspondingly different times after the mixing-induced inception of protein folding. This flow/rapid scan method is a means for monitoring early immobilization of the spin probe in the course of the folding process.

Topics: Antioxidants; Cytochrome c Group; Cytochromes c; Dehydroascorbic Acid; Electron Spin Resonance Spectroscopy; Free Radicals; Magnetics; Nitrates; Nitrogen Oxides; Oxidants; Protein Folding; Pulsatile Flow; Saccharomyces cerevisiae Proteins; Spin Labels