Presence Of Ascorbate Research Articles

Ascorbate: a forgotten component in the cytotoxicity of Cu(II) ATCUN peptide complexes.

In 1983, Linus Pauling and colleagues reported about enhanced antitumor activity of the Cu(II) complex of the simplest ATCUN (amino terminal Cu(II) and Ni(II)-binding motif) peptide (NH2-Gly-Gly-His-COOH, GGH) in the presence of ascorbate as an additive. In the following 4 decades, structural modifications of this complex were implemented, however, anticancer activity could not be significantly increased. This has led to neglecting the ATCUN motif and its Cu(II) complexes as potential chemotherapeutic agents. Furthermore, the addition of ascorbate with its positive effect on the anticancer activity has fallen into oblivion. In this work, we compared Cu(II) GGH with Cu(II) ATCUN peptides bearing β-Ala instead of Gly at the 2nd position of the peptide sequence regarding their in vitro complex stability and cytotoxicity (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and annexin V-FITC (fluorescein isothiocyanate) apoptosis assay) towards three cancer cell lines (AGS, HeLa and NCI-N87). Such an exchange of amino acids led to an up to three-fold higher cytotoxic effect in the presence of ascorbate. We thus achieved a significant increase in the otherwise moderate cytotoxicity of Cu(II) ATCUN-like complexes. Lipophilicity assays (n-octanol/water coefficient, log P values) of the studied complexes were used to evaluate differences in the antiproliferative activity.

An unusual triplet population pathway in the Reaction Centre of the Chlorophyll-d binding Photosystem I of A. marina, as revealed by a combination of TR-EPR and ODMR spectroscopies

Photo-induced Chlorophyll (Chl) triplet states in the isolated Photosystem I (PSI) of Acaryochloris marina, that harbours Chl d as its main pigment, were investigated by Optically Detected Magnetic Resonance (ODMR) and Time-Resolved Electron Paramagnetic Resonance (TR-EPR), and as a function of pre-illumination of the sample under reducing redox poising. Fluorescence Detected Magnetic Resonance (FDMR) allowed resolving four Chl d triplet (3Chl d) populations (T1-T4) both in untreated and illuminated samples in the presence of ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The FDMR signals increased following the pre-illumination treatment, particularly for the T3 and T4 populations, which are therefore sensitive to the redox state of PSI cofactors. Microwave-induced Triplet minus Singlet (TmS) spectra were detected in the |D|-|E| resonance window of the T3 and T4 triplets. These showed a broad singlet bleaching centred at 740 nm and also displayed complex spectral structure with several derivative-like features, indicating that both the T3 and T43Chl d populations are associated with the PSI reaction centre (RC) triplet, P3740. Parallel measurements by TR-EPR demonstrated that triplet signals observed under all conditions investigated are dominated by an electron spin polarisation (esp), which is typical of intersystem crossing, differently from what expected for recombination triplet states formed from a radical pair precursor. Moreover, stronger reductant conditions obtained by pre-illumination of the samples in the presence of dithionite and 5-methylphenazinium methyl sulfate (PMS) did not lead to a recombination triplet state esp, but rather to a decrease of the whole signal intensity. The energetics of A. marina PSI and the possible occurrence of distributions of cofactors redox properties are discussed in order to address the unexpected P3740 esp.

A Metastable Semiquinone Molecular Switch Modulated by Ascorbate/O2: A Study from a System Far-From-Equilibrium to Biological Assays in Mitochondria.

A molecular switch based on the metastable radical anion derived from a substituted heteroaryl quinone is described. Pyrrolyl quinone thiocyanate (PQ 9) showed an interaction with the fluoride anion that was visible to the naked eye and quantified by UV/vis and 1H and 13 C NMR. The metastable quinoid species formed by the interaction with F- ("ON" state) showed a molecular switching effect autocontrolled by the presence of ascorbate ("OFF" state) and back to the "ON" state by an autooxidation process, measured by visible and UV/vis spectroscopy. Due to its out-of-equilibrium properties and the exchange of matter and energy, a dissipative structural behaviour is proposed. Considering its similarity to the mechanism of coenzyme Q in oxidative phosphophorylation, PQ 9 was evaluated on Saccharomyces cerevisiae mitochondrial function for inhibition of complexes II, III and IV, reactive oxygen species (ROS) production, catalase activity and lipid peroxidation. The results showed that PQ 9 inhibited complex III activity as well as the activity of all electron transport chain (ETC) complexes. In addition, PQ 9 reduced ROS production and catalase activity in yeast. The results suggest that PQ 9 may have potential applications as a new microbicidal compound by inducing ETC dysfunction.

Photo-induced imine reduction by a photoredox biocatalyst consisting of a pentapeptide and a Ru bipyridine terpyridine complex

Imine reduction is a useful reaction in the preparation of amine derivatives. Various catalysts have been reported to promote this reaction and photoredox catalysts are promising candidates for sustainable amine synthesis. Improvement of this reaction using biomolecule-based reaction scaffolds is expected to increase the utility of the reaction. In this context, we have recently investigated photoredox Ru complexes with pentapeptide scaffolds via coordination bonds as catalysts for photoreduction of dihydroisoquinoline derivatives. First, Ru bipyridine terpyridine complexes coordinated with five different pentapeptides (XVHVV: X = V, F, W, Y, C) were prepared and characterized by mass spectrometry. Catalytic activities of the Ru complexes with XVHVV were evaluated for photoreduction of dihydroisoquinoline derivatives in the presence of ascorbate and thiol compounds as sacrificial reagents and hydrogen sources. Interestingly, the turnover number of the Ru complex with VVHVV is 531, which is two-fold higher than that of a simple Ru complex with an imidazole ligand. The detailed emission lifetime measurements indicate that the enhanced catalytic activity provided by the peptide scaffold is caused by an efficient reaction with the thiol derivative to accelerate reductive quenching of Ru complex. The quenching behavior suggests formation of an active species such as a Ru(I) complex. These findings reveal that the simple pentapeptide serves as an effective scaffold to enhance the photocatalytic activity of a photoactive Ru complex.

Metabolomic Profiling and Characterization of a Novel 3D Culture System for Studying Chondrocyte Mechanotransduction.

Articular chondrocytes synthesize and maintain the avascular and aneural articular cartilage. In vivo these cells are surrounded by a 3D pericellular matrix (PCM) containing predominantly collagen VI. The PCM protects chondrocytes and facilitates mechanotransduction, and PCM stiffness is critical in transmitting biomechanical signals to chondrocytes. Various culture systems with different hydrogels have been used to encapsulate chondrocytes for 3D culture, but many lack either the PCM or the in vivo stiffness of the cartilage matrix. Here, we demonstrate that primary chondrocytes cultured in alginate will form a pericellular matrix and display a phenotype similar to in vivo conditions. We found that primary human and bovine chondrocytes, when cultured in alginate beads with addition of sodium L-ascorbate for 7 days, had a pronounced PCM, retained their phenotype, and synthesized both collagens VI and II. This novel culture system enables alginate-encapsulated chondrocytes to develop a robust PCM thereby creating a model system to study mechanotransduction. We also observed distinct compression-induced changes in metabolomic profiles between the monolayer-agarose and alginate-released agarose-embedded chondrocytes indicating physiological changes in cell metabolism. Our data suggest that 3D preculture of chondrocytes in alginate before encapsulation in physiologically-stiff agarose leads to a pronounced development of pericellular matrix that is sustained in the presence of ascorbate. This novel model can be useful in studying the mechanism by which chondrocytes respond to cyclical compression and other types of loading simulating in vivo physiological conditions.

Quest for a stable Cu-ligand complex with a high catalytic activity to produce reactive oxygen species.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed.

An engineered POSS drug delivery system for copper(II) anticancer metallodrugs in a selective application toward melanoma cells.

In this work, a polyhedral silsesquioxane (POSS) was used as an engineered drug delivery system for two oxindolimine-copper(II) anticancer complexes, [Cu(isaepy)]+ and [Cu(isapn)]+. The interest in hybrid POSS comes from the necessity of developing materials that can act as adjuvants to improve the cytotoxicity of non-soluble metallodrugs. Functionalization of POSS with a triazole ligand (POSS-atzac) permitted the anchorage of such copper complexes, producing hybrid materials with efficient cytotoxic effects. Structural and morphological characterizations of these copper-POSS systems were performed by using different techniques (IR, NMR, thermogravimetric analysis). A combination of continuous-wave (CW) and pulsed EPR (HYSCORE) spectroscopies conducted at the X-band have enabled the complete characterization of the coordination environment of the copper ion in the POSS-atzac matrix. Additionally, the cytotoxic effects of the loaded materials, [Cu(isapn)]@POSS-atzac and [Cu(isaepy)]@POSS-atzac, were assessed toward melanomas (SK-MEL), in comparison to non-tumorigenic cells (fibroblast P4). Evaluation of their nuclease activity or ability to facilitate cleavage of DNA indicated concentrations as low as 0.6 μg mL-1, while complete DNA fragmentation was observed at 25 μg mL-1. By using adequate scavengers, investigations on active intermediates responsible for their cytotoxicity were performed, both in the absence and in the presence of ascorbate as a reducing agent. Based on the observed selective cytotoxicity of these materials toward melanomas, investigations on the reactivity of these complexes and corresponding POSS-materials with melanin, a molecule that contributes to melanoma resistance to chemotherapy, were carried out. Results indicated the main role of the binuclear copper species, formed at the surface of the silica matrix, in the observed reactivity and selectivity of these copper-POSS systems.

Gastric stability of bare and chitosan-fabricated ferritin and its bio-mineral: implication for potential dietary iron supplements.

Iron deficiency anaemia (IDA), the most widespread nutritional disorder, is a persistent global health issue affecting millions, especially in resource-limited geographies. Oral iron supplementation is usually the first choice for exogenous iron administration owing to its convenience, effectiveness and low cost. However, commercially available iron supplementations are often associated with oxidative stress, gastrointestinal side effects, infections and solubility issues. Herein, we aim to address these limitations by employing ferritin proteins-self-assembled nanocaged architectures functioning as a soluble cellular iron repository-as a non-toxic and biocompatible alternative. Our in vitro studies based on PAGE and TEM indicate that bare ferritin proteins are resistant to gastric conditions but their cage integrity is compromised under longer incubation periods and at higher concentrations of pepsin, which is a critical component of gastric juice. To ensure the safe delivery of encapsulated iron cargo, with minimal cage disintegration/degradation and iron leakage along the gastrointestinal tract, we fabricated the surface of ferritin with chitosan. Further, the stoichiometry and absorptivity of iron-chelator complexes at both gastric and circumneutral pH were estimated using Job's plot. Unlike bipyridyl, deferiprone exhibited pH dependency. In vitro kinetics was studied to evaluate iron release from bare and chitosan-fabricated ferritins employing both reductive (in the presence of ascorbate and bipyridyl) and non-reductive (direct chelation by deferiprone) pathways to determine their bio-mineral stabilities. Chitosan-decorated ferritin displayed superior cage integrity and iron retention capability over bare ferritin in simulated gastric fluid. The ability of ferritins to naturally facilitate controlled iron release in conjugation with enteric coating provided by chitosan may mitigate the aforementioned side effects and enhance iron absorption in the intestine. The results of the current study could pave the way for the development of an oral formulation based on ferritin-caged iron bio-mineral that can be a promising alternative for the treatment of IDA, offering better therapeutic outcomes.

Anti-Proliferation and DNA Cleavage Activities of Copper(II) Complexes of N3O Tripodal Polyamine Ligands

Four ligands based on the 2-tert-butyl-4-X-6-{Bis[(6-methoxy-pyridin-2-ylmethyl)-amino]-methyl}-phenol unit are synthesized: X = CHO (HLCHO), putrescine-pyrene (HLpyr), putrescine (HLamine), and 2-tert-butyl-4-putrescine-6-{Bis[(6-methoxy-pyridin-2-ylmethyl)-amino]-methyl}-phenol (H2Lbis). Complexes 1, 2, 3, and 4 are formed upon chelation to copper(II). The crystal structure of complex 1 shows a square pyramidal copper center with a very weakly bound methoxypridine moiety in the apical position. The pKa of the phenol moiety is determined spectrophotometrically at 2.82–4.39. All the complexes show a metal-centered reduction in their CV at Epc,red = −0.45 to −0.5 V vs. SCE. The copper complexes are efficient nucleases towards the ϕX174 DNA plasmid in the presence of ascorbate. The corresponding IC50 value reaches 7 μM for 2, with a nuclease activity that follows the trend: 2 > 3 > 1. Strand scission is promoted by the hydroxyl radical. The cytotoxicity is evaluated on bladder cancer cell lines sensitive (RT112) or resistant to cisplatin (RT112 CP). The IC50 of the most active complexes (2 and 4) is 1.2 and 1.0 μM, respectively, for the RT112 CP line, which is much lower than cisplatin (23.8 μM).

The precipitation process for a rare earth leach solution with calcium oxide in the presence of ascorbate and calcination to oxide of high purity

Sulfate ions coprecipitate with rare earth metal ions to form alkaline rare earth sulfate (RE2(OH)4SO4) in the CaO precipitation process, leading to excessive content of sulfate in the mixed rare earth oxide. Due to the difference of coordination ability between ascorbate, sulfate, hydroxyl and rare earth metal ions, ascorbate is introduced into the pregnant leach solution (PLS) during the CaO precipitation process. It realizes the coprecipitation between ascorbate (C6H7O6−) and rare earth metal ions, reducing the formation of RE2(OH)4SO4 and improving the purity of mixed rare earth oxide. In this paper, the standard reaction Gibbs free energy changes of the possible reactions in the precipitation process were calculated using the group contribution method, to draw the species distribution equilibrium diagram with Newton's iteration method. When ascorbate exists, rare earth metal ions in the PLS tend to form the alkaline rare earth ascorbate (RE2(OH)4(C6H7O6)2) instead of RE2(OH)4SO4. Furthermore, the effect of the reaction conditions on the precipitation process were studied. When the molar ratio of rare earth metal ions to ascorbate was 1.17, the purity of the mixed rare earth oxide was 96.2 wt% and the sulfate content was 2.2 wt%. Finally, based on the results of the material balance calculation, FTIR, XRD and TG-DSC tests, it was further confirmed that ascorbate participated in the form of RE2(OH)4(C6H7O6)2, and effectively suppressed the generation of RE2(OH)4SO4. Consequently, the results facilitate the green and efficient extraction of rare earths from the ion-adsorption type ore.

Heme binding to the SARS-CoV-2 spike glycoprotein

The target for humoral immunity, SARS-CoV-2 spike glycoprotein, has become the focus of vaccine research and development. Previous work demonstrated that the N-terminal domain (NTD) of SARS-CoV-2 spike binds biliverdin-a product of heme catabolism-causing a strong allosteric effect on the activity of a subset of neutralizing antibodies. Herein, we show that the spike glycoprotein is also able to bind heme (KD= 0.5± 0.2μM). Molecular modeling indicated that the heme group fits well within the same pocket on the SARS-CoV-2 spike NTD. Lined by aromatic and hydrophobic residues (W104, V126, I129, F192, F194, I203, and L226), the pocket provides a suitable environment to stabilize the hydrophobic heme. Mutagenesis of N121 has a substantive effect on heme binding (KD= 3000± 220μM), confirming the pocket as a major heme binding location of the viral glycoprotein. Coupled oxidation experiments in the presence of ascorbate indicated that the SARS-CoV-2 glycoprotein can catalyze the slow conversion of heme to biliverdin. The heme trapping and oxidation activities of the spike may allow the virus to reduce levels of free heme during infection to facilitate evasion of the adaptive and innate immunity.

Light-Driven Hydrogen Evolution Reaction Catalyzed by a Molybdenum-Copper Artificial Hydrogenase.

Orange protein (Orp) is a small bacterial metalloprotein of unknown function that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S2MoS2CuS2MoS2]3-. In this paper, the performance of Orp as a catalyst for the photocatalytic reduction of protons into H2 has been investigated under visible light irradiation. We report the complete biochemical and spectroscopic characterization of holo-Orp containing the [S2MoS2CuS2MoS2]3- cluster, with docking and molecular dynamics simulations suggesting a positively charged Arg, Lys-containing pocket as the binding site. Holo-Orp exhibits excellent photocatalytic activity, in the presence of ascorbate as the sacrificial electron donor and [Ru(bpy)3]Cl2 as the photosensitizer, for hydrogen evolution with a maximum turnover number of 890 after 4 h irradiation. Density functional theory (DFT) calculations were used to propose a consistent reaction mechanism in which the terminal sulfur atoms are playing a key role in promoting H2 formation. A series of dinuclear [S2MS2M'S2MS2](4n)- clusters, with M = MoVI, WVI and M'(n+) = CuI, FeI, NiI, CoI, ZnII, CdII were assembled in Orp, leading to different M/M'-Orp versions which are shown to display catalytic activity, with the Mo/Fe-Orp catalyst giving a remarkable turnover number (TON) of 1150 after 2.5 h reaction and an initial turnover frequency (TOF°) of 800 h-1 establishing a record among previously reported artificial hydrogenases.

Copper ions and the R peptides of tau: structural and functional study

AbstractBackgroundOne of the common neurodegenerative disorders is the aggregation of Tau protein plays a major role in Alzheimer’s disease. Transmission metals ions such as Cu, Zn, and Fe are vital ingredients that must maintain homeostasis in the biological system. Tau Protein has four repeat peptide units (R1 R2 R3 R4) which may coordinate divalent copper ions to form neurofibrillary tangles (NFTs) and generate reactive oxygen species (ROS).The abnormal amount of Cu2+ accumulation in the intracellular environment, regulating the biological systems is not clearly understood.MethodThe main objective of this study was to evaluate the coordination of four R peptides to Cu2+ by MALDI‐MS‐TOF and determine the interaction of Cu2+ ions with peptides (R1 R2 R3 R4) of Tau protein in vitro. Also, their role in the formation of ROS with a natural reducing agent‐ ascorbate was probed.ResultAs the principal findings of this experiment, metal ion coordinated all 4 R peptides, via His and Cys residue including N terminal and amide groups. All the metallo‐peptide complexes were singly charged peaks, and observed even in the presence of ascorbate. R2 and R3 peptides formed the homodimers by disulfide bond formation. The metallo‐peptides were antioxidants and prooxidants depending on the conditions, indicating that a complex behavior may exist in the biological setting.ConclusionThis work indicates that metal ions interact with R peptides, which may mitigate ROS levels. The finding of this study is greatly beneficial for understanding the Tau protein behavior to develop a potential medication.

Conversion of light into electricity in a semi-synthetic system based on photosynthetic bacterial chromatophores

Chromatophores (Chr) from photosynthetic nonsulfur purple bacterium Rhodobacter sphaeroides immobilized onto a Millipore membrane filter (MF) and sandwiched between two semiconductor indium tin oxide (ITO) electrodes (termed ITO|Chr – MF|ITO) have been used to measure voltage (ΔV) induced by continuous illumination. The maximum ΔV was detected in the presence of ascorbate / N,N,N’N'-tetramethyl-p-phenylenediamine couple, coenzyme UQ0, disaccaride trehalose and antimycin A, an inhibitor of cytochrome bc1 complex. In doing so, the light-induced electron transfer in the reaction centers was the major source of photovoltages. The stability of the voltage signal upon prolonged irradiation (>1 h) may be due to the maintenance of a conformation that is optimal for the functioning of integral protein complexes and stabilization of lipid bilayer membranes in the presence of trehalose. Retaining ∼70 % of the original photovoltage performance on the 30th day of storage at 23 °C in the dark under air was achieved after re-injection of fresh buffer (∼40 μL) containing redox mediators into the ITO|Chr – MF|ITO system. The approach we use is easy and can be extended to other biological intact systems (cells, thylakoid membranes) capable of converting energy of light.

Regioselective protein oxidative cleavage enabled by enzyme-like recognition of an inorganic metal oxo cluster ligand

Oxidative modifications of proteins are key to many applications in biotechnology. Metal-catalyzed oxidation reactions efficiently oxidize proteins but with low selectivity, and are highly dependent on the protein surface residues to direct the reaction. Herein, we demonstrate that discrete inorganic ligands such as polyoxometalates enable an efficient and selective protein oxidative cleavage. In the presence of ascorbate (1 mM), the Cu-substituted polyoxometalate K8[Cu2+(H2O)(α2-P2W17O61)], (CuIIWD, 0.05 mM) selectively cleave hen egg white lysozyme under physiological conditions (pH =7.5, 37 °C) producing only four bands in the gel electropherogram (12.7, 11, 10, and 5 kDa). Liquid chromatography/mass spectrometry analysis reveals a regioselective cleavage in the vicinity of crystallographic CuIIWD/lysozyme interaction sites. Mechanistically, polyoxometalate is critical to position the Cu at the protein surface and limit the generation of oxidative species to the proximity of binding sites. Ultimately, this study outlines the potential of discrete, designable metal oxo clusters as catalysts for the selective modification of proteins through radical mechanisms under non-denaturing conditions.

Epigenetic Remodeling by Vitamin C Potentiates the Differentiation of Mouse and Human Plasma Cells

Abstract Ascorbate (vitamin C) is an essential micronutrient in humans. The chronic severe deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. Here, from a micronutrient screen, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique, as other antioxidants failed to promote plasma cell differentiation. Ascorbate is critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. Consistent with its role as a cofactor for epigenetic enzymes, ascorbate potentiates plasma cell differentiation by remodeling the epigenome via TET (Ten Eleven Translocation), the enzymes responsible for DNA demethylation by oxidizing 5-methylcytosines into 5-hydroxymethylcytosine (5hmC). Genome-wide 5hmC profiling identified ascorbate responsive elements (EAR) at the Prdm1 locus, including a distal element with a STAT3 motif overlapped with a CpG that was methylated and modified by TET in the presence of ascorbate. The results suggest that an adequate level of VC is required for antibody response and highlight how micronutrients regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions. This research was funded by LJI/KKR young investigator fund; NIH National Cancer Institute K22 (K22CA241290); startup funds from the Department of Microbial Infection and Immunity and from the Pelotonia Institute of Immuno-oncology at the Ohio State University.

Ascorbate-and iron-driven redox activity of Dp44mT and Emodin facilitates peroxidation of micelles and bicelles

BackgroundIron (Fe)-induced oxidative stress leads to reactive oxygen species that damage biomembranes, with this mechanism being involved in the activity of some anti-cancer chemotherapeutics. MethodsHerein, we compared the effect of the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on Fe-catalyzed lipid peroxidation in cell membrane models (micelles and bicelles). These studies were performed in the presence of hydrogen peroxide (H2O2) and the absence or presence of ascorbate. ResultsIn the absence of ascorbate, Fe(II)/Emodin mixtures incubated with H2O2 demonstrated slight pro-oxidant properties on micelles versus Fe(II) alone, while the Fe(III)-Dp44mT complex exhibited marked antioxidant properties. Examining more physiologically relevant phospholipid-containing bicelles, the Fe(II)- and Fe(III)-Dp44mT complexes demonstrated antioxidant activity without ascorbate. Upon adding ascorbate, there was a significant increase in the peroxidation of micelles and bicelles in the presence of unchelated Fe(II) and H2O2. The addition of ascorbate to Fe(III)-Dp44mT substantially increased the peroxidation of micelles and bicelles, with the Fe(III)-Dp44mT complex being reduced by ascorbate to the Fe(II) state, explaining the increased reactivity. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radical anion generation after mixing ascorbate and Emodin, with signal intensity being enhanced by H2O2. This finding suggested Emodin semiquinone radical formation that could play a role in its reactivity via ascorbate-driven redox cycling. Examining cultured melanoma cells in vitro, ascorbate at pharmacological levels enhanced the anti-proliferative activity of Dp44mT and Emodin. Conclusions and general significanceAscorbate-driven redox cycling of Dp44mT and Emodin promotes their anti-proliferative activity.

Glypican-3-Targeted 227Th α-Therapy Reduces Tumor Burden in an Orthotopic Xenograft Murine Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a significant cause of morbidity and mortality worldwide, with limited therapeutic options for advanced disease. Targeted α-therapy is an emerging class of targeted cancer therapy in which α-particle-emitting radionuclides, such as 227Th, are delivered specifically to cancer tissue. Glypican-3 (GPC3) is a cell surface glycoprotein highly expressed on HCC. In this study, we describe the development and invivo efficacy of a 227Th-labeled GPC3-targeting antibody conjugate (227Th-octapa-αGPC3) for treatment of HCC in an orthotopic murine model. Methods: The chelator p-SCN-Bn-H4octapa-NCS (octapa) was conjugated to a GPC3-targeting antibody (αGPC3) for subsequent 227Th radiolabeling (octapa-αGPC3). Conditions were varied to optimize radiolabeling of 227Th. In vitro stability was evaluated by measuring the percentage of protein-bound 227Th by γ-ray spectroscopy. An orthotopic athymic Nu/J murine model using HepG2-Red-FLuc cells was developed. Biodistribution and blood clearance of 227Th-octapa-αGPC3 were evaluated in tumor-bearing mice. The efficacy of 227Th-octapa-αGPC3 was assessed in tumor-bearing animals with serial measurement of serum α-fetoprotein at 23 d after injection. Results: Octapa-conjugated αGPC3 provided up to 70% 227Th labeling yield in 2 h at room temperature. In the presence of ascorbate, at least 97.8% of 227Th was bound to αGPC3-octapa after 14 d in phosphate-buffered saline. In HepG2-Red-FLuc tumor-bearing mice, highly specific GPC3 targeting was observed, with significant 227Th-octapa-αGPC3 accumulation in the tumor over time and minimal accumulation in normal tissue. Twenty-three days after treatment, a significant reduction in tumor burden was observed in mice receiving a 500 kBq/kg dose of 227Th-octapa-αGPC3 by tail-vein injection. No acute off-target toxicity was observed, and no animals died before termination of the study. Conclusion:227Th-octapa-αGPC3 was observed to be stable invitro; maintain high specificity for GPC3, with favorable biodistribution invivo; and result in significant antitumor activity without significant acute off-target toxicity in an orthotopic murine model of HCC.

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl3) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.

Microelectrode Sensor for Real-Time Measurements of Nitrite in the Living Brain, in the Presence of Ascorbate.

The impaired blood flow to the brain causes a decrease in the supply of oxygen that can result in cerebral ischemia; if the blood flow is not restored quickly, neuronal injury or death will occur. Under hypoxic conditions, the production of nitric oxide (●NO), via the classical L-arginine–●NO synthase pathway, is reduced, which can compromise ●NO-dependent vasodilation. However, the alternative nitrite (NO2−) reduction to ●NO, under neuronal hypoxia and ischemia conditions, has been viewed as an in vivo storage pool of ●NO, complementing its enzymatic synthesis. Brain research is thus demanding suitable tools to probe nitrite’s temporal and spatial dynamics in vivo. In this work, we propose a new method for the real-time measurement of nitrite concentration in the brain extracellular space, using fast-scan cyclic voltammetry (FSCV) and carbon microfiber electrodes as sensing probes. In this way, nitrite was detected anodically and in vitro, in the 5–500 µM range, in the presence of increasing physiological concentrations of ascorbate (100–500 µM). These sensors were then tested for real-time and in vivo recordings in the anesthetized rat hippocampus; using fast electrochemical techniques, local and reproducible transients of nitrite oxidation signals were observed, upon pressure ejection of an exogenous nitrite solution into the brain tissue. Nitrite microsensors are thus a valuable tool for investigating the role of this inorganic anion in brain redox signaling.