Effects of Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor and Human Parathyroid Hormone Fragment 13-34

No HeadingPurpose. The effects and mechanisms of different antioxidants, methionine, glutathione, acetylcysteine, and ascorbic acid (AscH₂), on the oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone fragment 13-34 (hPTH 13-34) by hydrogen peroxide (H₂O₂) were quantified and analyzed.Methods. The rates of oxidation of methionine residues in G-CSF were determined by peptide mapping analyses, and the oxidation of methionine residue in hPTH 13-34 was quantified by reverse-phase HPLC.Results. At pH 4.5, free methionine reduces, glutathione and acetylcysteine have no obvious effect on, and AscH₂ promotes the rates of oxidation of methionine residues in G-CSF. The H₂O₂-induced oxidation rate constants for free methionine, acetylcysteine, and glutathione at pH 4.5 were measured to be 32.07, 1.00, and 1.63 M^-1h^-1, respectively, while the oxidation rate constant for Met¹, the most readily oxidizable methionine residue in G-CSF, is 13.95 M^–1h^–1. Therefore, the different effects of free methionine, acetylcysteine, and glutathione on the rates of oxidation of methionine residues in G-CSF are consistent with their different reactivity toward oxidation by H₂O₂. By using hPTH 13-34, the effect of AscH₂ on the H₂O₂-induced oxidation of methionine residue was quantified, and the mechanisms involved were proposed. Because of the presence of trace transition metal ions in solution, at low concentrations, AscH₂ is prone to be a prooxidant, increasing the hydroxyl radical (OH) production rate via Fenton-type reactions. In addition to peroxide oxidation, these radicals lead to the degradation of hPTH 13-34 to smaller peptide fragments. At high concentrations, AscH₂ tends to act as an OH scavenger. EDTA inhibits OH production and thus eliminates the degradation of hPTH 13-34 by forming complexes with transition metal ions. However, the rate of oxidation of the methionine residue in hPTH 13-34 increases as the concentration of AscH₂ is increased from 0 to 200 mM, and the reason for this is still not clear.Conclusions. Our results demonstrate that free methionine is an effective antioxidant to protect G-CSF against methionine oxidation at pH 4.5. Acetylcysteine and glutathione are not effective antioxidants at pH 4.5. Their oxidation rates at different pH values imply that they would be much more effective antioxidants than free methionine at alkaline conditions. AscH₂ is a powerful electron donor. It acts as a prooxidant in the conditions in this study and is unlikely to prevent oxidation by H₂O₂ in protein formulation, whether or not EDTA is present.     

Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies

The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography–mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G–Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.     

Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ～200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.     

Prediction of the Hydrogen Peroxide–Induced Methionine Oxidation Propensity in Monoclonal Antibodies

Methionine oxidation in therapeutic antibodies can impact the product's stability, clinical efficacy, and safety and hence it is desirable to address the methionine oxidation liability during antibody discovery and development phase. Although the current experimental approaches can identify the oxidation-labile methionine residues, their application is limited mostly to the development phase. We demonstrate an in silico method that can be used to predict oxidation-labile residues based solely on the antibody sequence and structure information. Since antibody sequence information is available in the discovery phase, the in silico method can be applied very early on to identify the oxidation-labile methionine residues and subsequently address the oxidation liability. We believe that the in silico method for methionine oxidation liability assessment can aid in antibody discovery and development phase to address the liability in a more rational way.     

Effects of Excipients on the Hydrogen Peroxide-Induced Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor

No HeadingPurpose. The objective of this study was to elucidate the different mechanisms of action of different excipients on the oxidation of Met¹, Met¹²², Met¹²⁷, and Met¹³⁸ in granulocyte colony-stimulating factor (G-CSF) by using hydrogen peroxide as the oxidant.Methods. The oxidation of Met¹, Met¹²⁷, and Met¹³⁸ was quantified by peptide mapping analysis. The oxidation of Met¹²² has biphasic oxidation kinetics with a faster second phase. Therefore, the oxidation of Met¹²² was quantified by two different methods: peptide mapping analysis for the first phase of oxidation and direct reverse-phase HPLC for the second phase of oxidation.Results. The current work reveals that the preferential excluding excipients sorbitol, sucrose, and trehalose, in the concentration range 0–30% (w/v), and the preferential binding excipients urea and guanidine hydrochloride, in the concentration range 0–0.8 M, do not affect the oxidation of methionine residues in G-CSF at pH 4.5. The chelating agents citrate and EDTA have different effects on the rates of oxidation of methionine residues in G-CSF. At low concentrations, citrate decreases the rates, while at high concentrations, citrate increases the rates. EDTA decreases the rates of oxidation of methionine residues in G-CSF, such that its effect becomes more and more as its concentration is increased from 0 to 200 mM. The efficacy of EDTA on the rates of oxidation of the four methionine residues in G-CSF follows the order Met¹²² > Met¹²⁷ > Met¹³⁸ > Met¹.Conclusions. Our results indicate that EDTA can protect the methionine residues in G-CSF against oxidation induced by hydrogen peroxide. The more exposed the methionine residue is, the more difficult it is to be protected by EDTA. The mechanism may be due to the specific ion binding of EDTA to proteins.     

Enhanced physical stability of human calcitonin after methionine oxidation

Calcitonin is a blood-calcium-lowering peptide, present in different species, which inhibits the resorption of bone by osteoclasts. Human calcitonin (hCT) is one of the few calcitonin peptides, which contains a methionine residue; this residue is in position 8. Methionines are known to be readily oxidized to sulfoxides both in vivo and in vitro. The current work describes the effect of methionine oxidation on the physical stability of hCT. Aggregation kinetics of human calcitonin were studied at different pH values by intrinsic fluorescence spectroscopy, turbidity at 350 nm, microscopy analyses, Nile Red, and 1,8-ANS fluorescence emission. In all the experiments, methionine oxidation reduced the aggregation rate of human calcitonin. The effect of methionine oxidation was independent of pH. Fluorescence lifetime data also showed that the conformation of hCT in the aggregated state can be influenced by methionine oxidation. A hypothesis for the enhanced physical stability of oxidized hCT is presented and discussed.     

The role of formate and S-adenosylmethionine in the reversal of nitrous oxide inhibition of formate oxidation in the rat

Studies have been performed in rats in order to test whether methionine reverses the inhibition of formate oxidation produced by nitrous oxide by virtue of the conversion of methionine to formate. At a dose of methionine (100 mg/kg, 671 mumol/kg) that completely reverses the nitrous oxide inhibition of formate oxidation no significant conversion of the methyl group, carboxyl, or backbone of methionine to formate was apparent. No increases in hepatic formate levels were seen after the administration of 671 mumol/kg methionine or ethionine, and formate treatment did not alter the rate of 14CO2 formed after methionine was administered labeled in the methyl, carboxyl, or backbone position. The reversal of nitrous oxide inhibition of formate oxidation was found to correlate temporally with either S-adenosylmethionine levels after methionine administration or S-adenosylethionine levels following ethionine treatment. After methionine or ethionine administration, elevated hepatic steady state levels of tetrahydrofolate were observed and were coincident with elevated S-adenosylmethionine or S-adenosylethionine. Since formate oxidation rates are dependent on the hepatic tetrahydrofolate level, the mechanism of methionine reversal of nitrous oxide inhibition appears to be related to effects of hepatic S-adenosylmethionine which are important in maintaining and regulating tetrahydrofolate, rather than formate generation from methionine.     

Role of phenylalanine 20 in Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity

Senile plaques are a hallmark of Alzheimer's disease (AD), a neurodegenerative disease associated with cognitive decline and aging. Abeta(1-42) is the primary component of the senile plaque in AD brain and has been shown to induce protein oxidation in vitro and in vivo. Oxidative stress is extensive in AD brain. As a result, Abeta(1-42) has been proposed to play a central role in the pathogenesis of AD; however, the specific mechanism of neurotoxicity remains unknown. Recently, it has been proposed that long distance electron transfer from methionine 35 to the Cu(II) bound at the N terminus of Abeta(1-42) occurs via phenylalanine 20. Additionally, it was proposed that substitution of phenylalanine 20 of Abeta(1-42) by alanine [Abeta(1-42)F20A] would lessen the neurotoxicity induced by Abeta(1-42). In this study, we evaluate the predictions of this theoretical study by determining the oxidative stress and neurotoxic properties of Abeta(1-42)F20A relative to Abeta(1-42) in primary neuronal cell culture. Abeta(1-42)F20A induced protein oxidation and lipid peroxidation similar to Abeta(1-42) but to a lesser extent and in a manner inhibited by pretreatment of neurons with vitamin E. Additionally, Abeta(1-42)F20A affected mitochondrial function similar to Abeta(1-42), albeit to a lesser extent. Furthermore, the mutation does not appear to abolish the ability of the native peptide to reduce Cu(II). Abeta(1-42)F20A did not compromise neuronal morphology at 24 h incubation with neurons, but did so after 48 h incubation. Taken together, these results suggest that long distance electron transfer from methionine 35 through phenylalanine 20 may not play a pivotal role in Abeta(1-42)-mediated oxidative stress and neurotoxicity.     

Horseradish peroxidase-catalyzed oxidation of rifampicin: reaction rate enhancement by co-oxidation with anti-inflammatory drugs

The tuberculostatic drug rifampicin has been described as a scavenger of reactive species. Additionally, the recent demonstration that oral therapy with a complex of rifampicin and horseradish peroxidase (HRP) was more effective than rifampicin alone, in an animal model of experimental leprosy, suggested the importance of redox reactions involving rifampicin and their relevance to the mechanism of action. Hence, we studied the oxidation of rifampicin catalyzed by HRP, since this enzyme may represent the prototype of peroxidation-mediated reactions. We found that the antibiotic is efficiently oxidized and that rifampicin-quinone is the product, in a reaction dependent on both HRP and hydrogen peroxide. The steady-state kinetic constants Km(app) (101+/-23 micromol/l), Vmax(app) (0.78+/-0.09 micromol/l.s(-1)) and kcat (5.1+/-0.6 s(-1)) were measured (n=4). The reaction rate was increased by the addition of co-substrates such as tetramethylbenzidine, salicylic acid, 5-aminosalicylic acid and paracetamol. This effect was explained by invoking an electron-transfer mechanism by which these drugs acted as mediators of rifampicin oxidation. We suggested that this drug interaction might be important at the inflammatory site.     

Differentiating the biosynthesis of pseudomonic acids A and B

Pseudomonic acid A (1) has been the dominant commercial pseudomonate antibiotic produced by Pseudomonas fluorescens. In specific shaken flask conditions initial fermentation accumulation of 1 is followed by preferential accumulation of the 8-hydroxy derivative, pseudomonic acid B (2). Biosynthetic probing with a pulse of [1-14C] acetate or L-[methyl-14C] methionine at early, mid and late stages of the fermentation gave relative patterns of radioactivity in 1 and 2 that are inconsistent with an assumption that 2 arises by oxidation of 1, or that 1 is formed by reduction of 2. Since [methyl-14C] methionine only labels carbons in the 12-carbon part of the pseudomonate molecule that is thought to be an early biosynthetic moiety, the evidence from radiolabelling experiments implies that preferential early oxidation of this biosynthetic intermediate causes the pathway diversion to accumulate 2 instead of 1.     

Oxidative Degradation of Antiflammin 2

Purpose. To study the oxidation of the methionine residue of antiflammin 2 (HDMNKVLDL, AF2) as a function of pH, buffer concentration, ionic strength, and temperature using different concentrations of hydrogen peroxide and to determine the accessibility of methionine residue to oxidation. Methods. Reversed-phase high-performance liquid chromatography (RPHPLC) was used as the main analytical method in determining the oxidation rates of AF2. Calibration curves for AF2 and the oxidation product, methionine sulfoxide of AF2 (Met(O)-3-AF2), were constructed for each measurement using standard materials. Fast Atom Bombardment Mass Spectroscopy (FABMS) was used to characterize the product. Results. Met(O)-3-AF2 was the only oxidation product detected at pH 3.0 to 8.0. The oxidation rates were independent of buffer concentrations, ionic strength, and pH from 3.0 to 7.0. However, there was an acceleration of the rates at basic pHs, and small amounts of degradation products other than Met(O)-3-AF2 were observed in this alkaline region. Conclusions. Oxidation of methionine in AF2 does not cause the biological inactivation reported by other laboratories since this drug is relatively stable under neutral conditions in the absence of oxiding agent.     

Stability of human growth hormone: influence of methionine oxidation on thermal folding

Oxidation, particularly of methionine residues, is one of the major chemical degradations of proteins. In a previous publication we studied the conformation of recombinant human growth hormone (r-hGH) selectively oxidized at Met14 and Met125. Conformation of oxidized r-hGH was found not different from that of nonoxidized r-hGH. In this paper, the effect of methionine oxidation on the thermal stability of r-hGH folding was investigated. The thermally induced unfolding process of the oxidized and nonoxidized protein was measured by monitoring the circular dichroism signal at 220 nm. The melting temperatures (T(m)) of the oxidized and nonoxidized r-hGH forms were determined at different pHs and in the presence of salts often used in pharmaceutical formulations. The effect of the location of the oxidized Met residue in the protein and the percentage of oxidation were investigated. Our findings indicate that the monoxidation of the two most accessible methionine residues of r-hGH-Met14 and Met125 - has no effect on the protein conformation. However, oxidation of these residues to form sulfoxides does influence the thermal stability of the protein folding. The presence of the polar oxygen atom on the methionine sulfoxide group thermally destabilizes r-hGH folding. The effect (T(m)) depends upon pH, ionic strength, and the location of the oxidized methionine residues in the protein. The thermal melting of r-hGH and its oxidized products is a highly cooperative process. Methionine oxidation leads to a thermal destabilization of the whole protein folding and is not just a local destabilization.     

Middle-Down Fragmentation for the Identification and Quantitation of Site-Specific Methionine Oxidation in an IgG1 Molecule

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle- down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.     

Functional consequences of methionine oxidation of hERG potassium channels

Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in human ether à go-go related gene 1 (hERG1), which encodes cardiac I(Kr) channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300 microM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to -60 or -100 mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30 mV but accelerated during depolarization to 0 or -10mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel.     

The Kinetics of Relaxin Oxidation by Hydrogen Peroxide

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10–14 kcal mol^–1, with an entropy of activation of the order of –30 cal K^–1 mol^–1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.     

Chemical Pathways of Peptide Degradation. VIII. Oxidation of Methionine in Small Model Peptides by Prooxidant/Transition Metal Ion Systems: Influence of Selective Scavengers for Reactive Oxygen Intermediates

In the presence of oxygen, Fe(III), and an appropriate electron donor (e.g. ascorbic acid, dithiothreitol), the oxidation of methionine residues to methionine sulfoxides in small model peptides can be induced. It is shown in this study that these oxidations can be retarded by catalase in a pH-dependent manner, by some hydroxyl radical scavengers, and by azide. In contrast, superoxide dismutase has only a minimal effect, indicating that the superoxide radical does not contribute significantly to the oxidation of the methionine residue. The experimental results can be interpreted by invoking hydrogen peroxide as the major oxidizing species at pH 7, whereas the involvement of free hydroxyl radicals seems to be negligible. Other reactive oxygen intermediates such as iron-bound hydroperoxy, or site-specifically generated reactive oxygen species may be actively involved in the oxidation of methionine residues at pH > 7.     

Effects of sucrose on rFVIIa aggregation and methionine oxidation

The aim of this study was to characterize the effects of sucrose on the stability of recombinant factor VIIa (rFVIIa), with special emphasis on aggregation and methionine oxidation, as well as to investigate the impact of various environmental conditions on the rFVIIa conformation. The stability of rFVIIa was studied at pH 5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas formation of methionine oxidation products was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy were used to study protein conformation. Stability studies showed that increasing sucrose concentrations reduced the loss of monomeric rFVIIa, and decreased formation of dimeric/oligomeric and polymeric rFVIIa. Preferential exclusion of the sugar from the protein’s surface, which shifts the protein molecular population away from expanded aggregation competent species and toward the compact native state, is thought to account for these observations. rFVIIa is sensitive to methionine oxidation; two mono-oxidized and one di-oxidized product were formed upon incubation. Unlike aggregation, methionine oxidation was found to increase in the presence of sucrose. The two methionine residues susceptible to oxidation are presumably located at the protein surface, and the chemical potential increase in the presence of sucrose may account for the increase in oxidation rate. While FTIR spectroscopy suggested that sucrose induces small conformational changes in the rFVIIa structure, CD spectroscopy did not support this finding. The secondary structure of precipitated rFVIIa was changed when compared to the native solution secondary structure. Appearance of bands characteristic of intermolecular β-sheet structure were found coincident with a decrease in -helix and intramolecular β-sheet structure.     

Isoniazid as a substrate and inhibitor of myeloperoxidase: Identification of amine adducts and the influence of superoxide dismutase on their formation

Neutrophils ingest Mycobacteria tuberculosis (Mtb) in the lungs of infected individuals. During phagocytosis they use myeloperoxidase (MPO) to catalyze production of hypochlorous acid (HOCl), their most potent antimicrobial agent. Isoniazid (INH), the foremost antibiotic in the treatment of tuberculosis, is oxidized by MPO. It rapidly reduced compound I of MPO [k=(1.22±0.05)×10(6)M(-1)s(-1)] but reacted less favorably with compound II [(9.8±0.6)×10(2)M(-1)s(-1)]. Oxidation of INH by MPO and hydrogen peroxide was unaffected by chloride, the physiological substrate for compound I, and the enzyme was partially converted to compound III. This indicates that INH is oxidized outside the classical peroxidation cycle. In combination with superoxide dismutase (SOD), MPO oxidized INH without exogenous hydrogen peroxide. SOD must favor reduction of oxygen by the INH radical to give superoxide and ultimately hydrogen peroxide. In both oxidation systems, an adduct with methionine was formed and it was a major product with MPO and SOD. We show that it is a conjugate of an acyldiimide with amines. INH substantially inhibited HOCl production by MPO and neutrophils below pharmacological concentrations. The reversible inhibition is explained by diversion of MPO to its ferrous and compound III forms during oxidation of INH. MPO, along with SOD released by Mtb, will oxidize INH at sites of infection and their interactions are likely to limit the efficacy of the drug, promote adverse drug reactions via formation of protein adducts, and impair a major bacterial killing mechanism of neutrophils.     

Methionine dependence of tumours: a biochemical strategy for optimizing paclitaxel chemosensitivity in vitro

Methionine dependence is a unique feature of cancer cells characterized by growth and cell cycle arrest (typically in S and G2/M) under conditions of methionine depletion. Following replenishment of media with methionine, the cell cycle blockade is reversible and during this recovery period, cells may become more susceptible to the action of cell cycle specific drugs. The response of a panel of methionine dependent (HTC, Phi-1, PC3 and 3T3) cells to vinblastine and paclitaxel was compared to methionine independent Hs-27 cells under conditions of methionine depletion (M-H+; methionine depleted media supplemented with homocysteine) and starvation (M-H-; media without methionine or homocysteine). All cell lines were significantly more resistant to both agents under M-H+ and M-H- conditions compared to controls under normal culture conditions [M+H-]; however, the magnitude of resistance was reduced in the methionine independent Hs-27 cells. During recovery from methionine depletion and starvation, the response of the methionine dependent cells to vinblastine and paclitaxel was significantly enhanced compared to controls. Although the activity of vinblastine on the Hs-27 cell line was comparable to controls, these methionine independent cells became significantly more resistant to paclitaxel during recovery studies (IC50 = 2.13 +/- 0.5 microM) compared to control cultures (IC50 = 0.13 +/- 0.15 microM). Whilst the mechanism responsible for this remains uncertain, the increased activity of paclitaxel against methionine dependent cells in conjunction with the decreased activity against Hs-27 cells suggests that methionine depletion strategies may enhance the therapeutic index of paclitaxel.     

Homolytic pathways drive peroxynitrite-dependent Trolox C oxidation

Peroxynitrite is a powerful oxidant implicated as a mediator in nitric oxide ((*)NO)- and superoxide (O(2)(*)(-))-dependent toxicity. Peroxynitrite homolyzes after (i) protonation, yielding hydroxyl ((*)OH) and nitrogen dioxide ((*)NO(2)) free radicals, and (ii) reaction with carbon dioxide (CO(2)), yielding carbonate radical anion (CO(3)(*)(-)) and (*)NO(2). Additionally, peroxynitrite reacts directly with several biomolecules. It is currently accepted that alpha-tocopherol is one important antioxidant in lipid compartments and its reactions with peroxynitrite or peroxynitrite-derived radicals may be relevant in vivo. Previous reports on the peroxynitrite reaction with Trolox C (TxOH)--an alpha-tocopherol water soluble analogue--suggested a direct and fast reaction. This was unexpected to us as judged from the known reactivities of peroxynitrite with other phenolic compounds; thus, we thoroughly investigated the kinetics and mechanism of the reaction of peroxynitrite with TxOH and its modulation by CO(2). Direct electron paramagnetic resonance studies revealed that Trolox C phenoxyl radical (TxO(*)) was the only paramagnetic species detected either in the absence or in the presence of CO(2). Stopped-flow spectrophotometry experiments revealed a sequential reaction mechanism, with the intermediacy of TxO(*) and the production of Trolox C quinone (TxQ). Reactions were zero-order with respect to TxOH and first-order in peroxynitrite and CO(2), demonstrating that the reaction of peroxynitrite with TxOH is indirect. In agreement, TxOH was unable to inhibit the direct peroxynitrite-mediated oxidation of methionine to methionine sulfoxide. TxOH oxidation yields to TxO(*) and TxQ with respect to peroxynitrite were approximately 60 and approximately 31%, respectively, and increased to approximately 73 and approximately 40%, respectively, in the presence of CO(2). At peroxynitrite excess over TxOH, the kinetics and mechanism of oxidation are more complex and involve the reactions of CO(3)(*)(-) with TxO(*) and the possible intermediacy of unstable NO(2)-TxOH adducts. Taken together, our results strongly support that H(+)- or CO(2)-catalyzed homolysis of peroxynitrite is required to cause TxOH, and hence, alpha-tocopherol oxidation.