Extracellular Redox State Research Articles

Nanomaterials-Induced Redox Imbalance: Challenged and Opportunities for Nanomaterials in Cancer Therapy.

Cancer cells typically display redox imbalance compared with normal cells due to increased metabolic rate, accumulated mitochondrial dysfunction, elevated cell signaling, and accelerated peroxisomal activities. This redox imbalance may regulate gene expression, alter protein stability, and modulate existing cellular programs, resulting in inefficient treatment modalities. Therapeutic strategies targeting intra- or extracellular redox states of cancer cells at varying state of progression may trigger programmed cell death if exceeded a certain threshold, enabling therapeutic selectivity and overcoming cancer resistance to radiotherapy and chemotherapy. Nanotechnology provides new opportunities for modulating redox state in cancer cells due to their excellent designability and high reactivity. Various nanomaterials are widely researched to enhance highly reactive substances (free radicals) production, disrupt the endogenous antioxidant defense systems, or both. Here, the physiological features of redox imbalance in cancer cells are described and the challenges in modulating redox state in cancer cells are illustrated. Then, nanomaterials that regulate redox imbalance are classified and elaborated upon based on their ability to target redox regulations. Finally, the future perspectives in this field are proposed. It is hoped this review provides guidance for the design of nanomaterials-based approaches involving modulating intra- or extracellular redox states for cancer therapy, especially for cancers resistant to radiotherapy or chemotherapy, etc.

Disruption of extracellular redox balance drives persistent lung fibrosis and impairs fibrosis resolution

Lung fibrosis is a devastating outcome of various diffuse parenchymal lung diseases. Despite rigorous research efforts, the mechanisms that propagate its progressive and nonresolving nature remain enigmatic. Oxidative stress has been implicated in the pathogenesis of lung fibrosis. However, the role of extracellular redox state in disease progression and resolution remains largely unexplored. Here, we show that compartmentalized control over extracellular reactive oxygen species (ROS) by aerosolized delivery of recombinant extracellular superoxide dismutase (ECSOD) suppresses an established bleomycin-induced fibrotic process in mice. Further analysis of publicly available microarray, RNA-seq and single-cell RNAseq datasets reveals a significant decrease in ECSOD expression in fibrotic lung tissues that can be spontaneously restored during fibrosis resolution. Therefore, we investigate the effect of siRNA-mediated ECSOD depletion during the established fibrotic phase on the self-limiting nature of the bleomycin mouse model. Our results demonstrate that in vivo knockdown of ECSOD in mouse fibrotic lungs impairs fibrosis resolution. Mechanistically, we demonstrate that transforming growth factor (TGF)-β1 downregulates endogenous ECSOD expression, leading to the accumulation of extracellular superoxide via Smad-mediated signaling and the activation of additional stores of latent TGF-β1. In addition, depletion of endogenous ECSOD during the fibrotic phase in the bleomycin model induces an apoptosis-resistant phenotype in lung fibroblasts through unrestricted Akt signaling. Taken together, our data strongly support the critical role of extracellular redox state in fibrosis persistence and resolution. Based on these findings, we propose that compartment-specific control over extracellular ROS may be a potential therapeutic strategy for managing fibrotic lung disorders.

Cerium Oxide Nanoparticle Administration to Skeletal Muscle Cells under Different Gravity and Radiation Conditions.

For their remarkable biomimetic properties implying strong modulation of the intracellular and extracellular redox state, cerium oxide nanoparticles (also termed “nanoceria”) were hypothesized to exert a protective role against oxidative stress associated with the harsh environmental conditions of spaceflight, characterized by microgravity and highly energetic radiations. Nanoparticles were supplied to proliferating C2C12 mouse skeletal muscle cells under different gravity and radiation levels. Biological responses were thus investigated at a transcriptional level by RNA next-generation sequencing. Lists of differentially expressed genes (DEGs) were generated and intersected by taking into consideration relevant comparisons, which led to the observation of prevailing effects of the space environment over those induced by nanoceria. In space, upregulation of transcription was slightly preponderant over downregulation, implying involvement of intracellular compartments, with the majority of DEGs consistently over- or under-expressed whenever present. Cosmic radiations regulated a higher number of DEGs than microgravity and seemed to promote increased cellular catabolism. By taking into consideration space physical stressors alone, microgravity and cosmic radiations appeared to have opposite effects at transcriptional levels despite partial sharing of molecular pathways. Interestingly, gene ontology denoted some enrichment in terms related to vision, when only effects of radiations were assessed. The transcriptional regulation of mitochondrial uncoupling protein 2 in space-relevant samples suggests perturbation of the intracellular redox homeostasis, and leaves open opportunities for antioxidant treatment for oxidative stress reduction in harsh environments.

Extracellular redox state shift: A novel approach to target prostate cancer invasion.

Extracellular superoxide dismutase (ECSOD) and the cysteine/glutamate transporter (Cys)/(xCT) are tumor microenvironment (TME) redox state homeostasis regulators. Altered expression of ECSOD and xCT can lead to imbalance of the TME redox state and likely have a profound effect on cancer invasion. In the present study, we investigated whether ECSOD and xCT could be therapeutic targets for prostate cancer (PCa) invasion. Immunohistochemistry of tumor microarray PCa tissues (N = 165) with high Gleason scores indicated that xCT protein expression is significantly increased while ECSOD protein expression is significantly decreased. Metastatic PCa indicated ECSOD protein expression is significantly decreased in epithelial area whereas xCT protein expression is significantly increased in stromal area. Furthermore, inhibition of extracellular O2•- by overexpression of ECSOD or alteration of the extracellular Cys/CySS ratio by knockdown of xCT protein inhibited PCa cell invasion. Simultaneous overexpression of ECSOD and knockdown xCT inhibited PCa cell invasion more than overexpression of ECSOD or knockdown of xCT alone. In the co-culturing system, simultaneous overexpression of ECSOD and knockdown of xCT in prostate stromal WPMY-1 cells inhibited PCa cell invasiveness more than overexpression of ECSOD alone. The decrease in PCa invasion correlated with increased of extracellular H2O2 levels. Notably, overexpression of catalase in TME reversed the inhibitory effect of ECSOD on cancer cell invasion. Impaired ECSOD activity and an upregulated of xCT protein expression may be clinical features of an aggressive PCa, particularly metastatic cancers and/or those with a high Gleason score. Therefore, shifting the extracellular redox state toward an oxidizing status by targeted modulation of ECSOD and xCT, in both cancer and stromal cells, may provide a greater strategy for potential therapeutic interventions of aggressive PCa.

Kv1.2 Channels at the Interface of Redox and Electrical Excitability

Kv1.2 is a prominent neuronal potassium channel subtype linked to severe epilepsies and movement disorders. We have demonstrated that neuronal and heterologously-expressed Kv1.2 channels exhibit use-dependent activation, a behavior characterized by progressive potentiation of channel activity during trains of repetitive stimuli. Use-dependent activation arises from a gating mode shift from a ‘reluctant’ slowly-activating mode to a ‘willing’ rapidly-activating mode with hyperpolarized voltage-dependence of activation. Moreover, Kv1.2 subunits recruit this use-dependent phenotype to heteromeric channel complexes. In this study, we demonstrate that use-dependent activation of Kv1.2 channel complexes is strongly regulated by a variety of exogenous and physiological redox species. Under ambient redox conditions, Kv1.2 channels exhibit marked cell-to-cell variability of use-dependent activation. However, exposure to mild reducing conditions normalizes this response, such that a pronounced use-dependent phenotype is consistently observed, together with a dramatic depolarizing shift of voltage-dependent activation with a V1/2 of 51+/- 6 mV. Mutagenesis of candidate cysteine residues in Kv1.2 did not affect redox sensitivity, therefore we hypothesize a role for an extrinsic redox-sensitive interacting partner. Using a variety of redox buffers, we demonstrate that use-dependent activation is steeply regulated by redox potentials between −50 and −100 mV, within the typical extracellular range. Furthermore, effects of membrane-impermeable reducing agents demonstrate that use-dependent activation is regulated by the extracellular redox state. Taken together, these findings suggest that Kv1.2 is a unique transducer of the extracellular environment, and may translate altered extracellular redox conditions to changes in cellular electrical function.

266 - Alteration of Tumor Microenvironmental Redox State Inhibits Prostate Cancer Metastasis

Cancer cells exhibit a plasticity of redox state so that the conversion between redox status in tumor microenvironment (TME) can be accomplished rapidly, thus, extracellular redox-related proteins may be a new potential therapeutic targets for metastasis. Cysteine(CyS)/glutamate transporter(xCT) and extracellular superoxide dismutase (ECSOD) are mainly TME-redox state hemeostasis regulator, one control redox coupling ratio CyS/Cystine(CySS), the latter control prooxidants level. Herein, we demonstrated xCT protein expression is significantly increased and correlated with high Gleason score PCa tissues. In contrast, ECSOD protein expression is significantly decreased in high Gleason score PCa tissues. A decrease in ECSOD may result in elevation of extracellular prooxidant levels due to the increase in O2●– and OONO●, while overexpression of xCT may result in imbalance of extracellular thiol couples. These changes in the extracellular redox state are likely to have a profound effect on activation of invasion-related proteins. Inhibition of extracellular O2●– by overexpression of ECSOD using adenovirus SOD3 transduction or altering extracellular Cys/CySS levels by knock-downed xCT protein expression using siRNA xCT resulted in inhibition of PCa cell invasion and decreased activity of MMP2. Simultaneously overexpressed ECSOD and knock-downed xCT inhibited PCa cell invasion greater than overexpressed ECSOD or knock-downed xCT alone (~70%, 50%, and 30%, respectively). In the co-culturing system, overexpressed ECSOD and knock-downed xCT in both PC3 and stromal cells (WPMY-1) decreased PCa cell invasion greater than overexpressed ECSOD and knock-downed xCT in only PC3 cells or stromal cells. In conclusion, our data indicated that synergic modulation of ECSOD and xCT expression would elevate the success of site- specific redox based therapies at TME. Since, paracrine signaling from stromal cells induce PCa cells to grow and metastatic, targeting ECSOD and xCT in both cancer and stromal cells would provide a greater inhibition of PCa metastasis.

Loss of Mrp1 Potentiates Doxorubicin-Induced Cytotoxicity in Neonatal Mouse Cardiomyocytes and Cardiac Fibroblasts.

Doxorubicin (DOX) induces dose-dependent cardiotoxicity in part due to its ability to induce oxidative stress. We showed that loss of multidrug resistance-associated protein 1 (Abcc1/Mrp1) potentiates DOX-induced cardiac dysfunction in mice in vivo Here, we characterized DOX toxicity in cultured cardiomyocytes (CM) and cardiac fibroblasts (CF) derived from C57BL wild type (WT) and Mrp1 null (Mrp1-/-) neonatal mice. CM accumulated more intracellular DOX relative to CF but this accumulation did not differ between genotypes. Following DOX (0.3-4 μM), Mrp1-/- CM, and CF, especially CM, showed a greater decrease in viability and increased apoptosis and DNA damage, demonstrated by higher caspase 3 cleavage, poly (ADP-ribose) polymerase 1 (PARP) cleavage and phosphorylated histone H2AX (γH2AX) levels versus WT cells. Saline- and DOX-treated Mrp1-/- cells had significantly higher intracellular GSH and GSSG compared with WT cells (P < .05), but the redox potential (Eh) of the GSH/GSSG pool did not differ between genotypes in CM and CF, indicating that Mrp1-/- cells maintain this major redox couple. DOX increased expression of the rate-limiting GSH synthesis enzyme glutamate-cysteine ligase catalytic (GCLc) and regulatory subunits (GCLm) to a significantly greater extent in Mrp1-/- versus WT cells, suggesting adaptive responses to oxidative stress in Mrp1-/- cells that were inadequate to afford protection. Expression of extracellular superoxide dismutase (ECSOD/SOD3) was lower (P < .05) in Mrp1-/- versus WT CM treated with saline (62% ± 8% of WT) or DOX (43% ± 12% of WT). Thus, Mrp1 protects CM in particular and CF against DOX-induced toxicity, potentially by regulating extracellular redox states.

The Intra- or Extracellular Redox State Was Not Affected by a High vs. Low Glycemic Response Diet in Mice.

A low glycemic response (LGR) vs. high glycemic response (HGR) diet helps curtail the development of obesity and diabetes, though the mechanisms are unknown. We hypothesized that consumption of a HGR vs. a LGR diet would lead to a more oxidized circulating redox state and predicted that a HGR diet would increase fat accumulation, reduce insulin sensitivity, and impair metabolic acclimation to a high fat diet in a mouse model. Hence, male C57BL/6 mice consumed a HGR or LGR diet for 16 weeks and a subset of the mice subsequently consumed a high fat diet for 4 weeks. We found that body mass increased at a faster rate for those consuming the HGR diet. Percent body fat was greater and percent lean mass was lesser in the HGR group starting at 12 weeks. However, the groups did not differ in terms of glucose tolerance at week 14 and metabolic parameters (respiratory exchange ratio, heat production, activity) at weeks 4 or 15. Moreover, mice on either diet did not show differences in metabolic acclimation to the high fat leg of the study. At the termination of the study, the groups did not differ in terms of redox pairs (lactate/pyruvate and β-hydroxybutyrate/acetoacetate) or thioredoxin reductase activity in blood. Also, total and oxidized glutathione levels and lipid peroxidation were similar in blood and liver. Correlations between baseline measures, longitudinal parameters, environmental conditions, and terminal metrics revealed that individual mice have innate propensities to metabolic regulation that may be difficult to perturb with diet alone; for example, starting mass correlated negatively with energy expenditure 4 weeks into the study and total hepatic glutathione at the end of the study. In conclusion, these data suggest that the mechanism by which HGR carbohydrates contributes to obesity is not via prolonged oxidation of the circulating redox state.

The Extracellular Redox State Modulates Mitochondrial Function, Gluconeogenesis, and Glycogen Synthesis in Murine Hepatocytes

Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB)/acetoacetate (Acoc), reduced glutathione (GSH)/oxidized glutathione (GSSG), and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(P)H and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

Fabrication of new single cell chip to monitor intracellular and extracellular redox state based on spectroelectrochemical method

Probing the local environment of target cells has been considered a challenging task due to the complexity of living cells. Here, we developed new single cell-based chip to investigate the intracellular and extracellular redox state of PC12 cells using spectroelectrochemical tool that combined surface-enhanced Raman scattering (SERS) and linear sweep voltammetry (LSV) techniques. PC12 cells immobilized on gold nanodots/ITO surface were subjected to LSV and their intracellular biochemical changes were successfully monitored by SERS simultaneously. Moreover, paired gold microelectrodes with micrometer-sized gap containing hexagonal array of gold nanodots were fabricated to detect electrochemical activity and changes in the redox environment of single PC12 cell based on SERS–LSV tool. This showed very effective detecting method. The used technology included the utilization of gold nanodots array inside micro-gap to enhance the Raman signals and the electrochemical activity of single cell. This could be used as an effective research tool to analyze cellular processes.

Selenitetriglycerides—Redox-active agents

BackgroundHuman prostate cancer (hPCa) is the most commonly diagnosed cancer in elderly men and is the second leading cause of male cancer death. Data from epidemiological, eco-environmental, nutritional prevention and clinical trials suggest that selenium Se(IV) can prevent prostate cancer. Selol, a new organic semisynthetic derivative of Se(IV), is a mixture of selenitetriglycerides. This mixture is non-toxic and non-mutagenic, and after po treatment – 56-times less toxic (in mice) than sodium selenite. It exhibits strong anti-cancer activity in vitro in many cancer cell lines and can overcome the cell resistance to doxorubicin. Selol seems a promising compound for prostate cancer therapy. Materials and methodsThe aim of the present study is the evaluation of Selol's influence on intracellular redox state (Eh) of prostatic tumors and the liver in androgen-dependent hPCa-bearing mice, and extracellular redox state in serum of these mice. Results and conclusionsThe anticancer activity of Selol involves perturbation of the redox regulation in the androgen dependent hPCa (LNCaP) cells, but not in healthy cells. After Selol treatment, intracellular Eh has increased in tumors from −223mV to −175mV, while in serum it has decreased (−82mV vs −113mV). It shows significant changes Eh in the extra- and intracellular environment. The difference decreases from 141mV to 62mV. The changes suggest that a tumor cell was probably directed toward apoptosis. This is exemplified in a significant decrease in cancer tumor mass by approx. 17% after the three weeks of Selol administration.

Extracellular redox state regulates catecholamine biosynthesis in PC12 cells with insulin resistance.

Extracellular cysteine (Cys)/cystine (CySS) redox potential (Eh) plays a crucial role in maintaining redox homeostasis and an alteration of redox state occurs in various physiological conditions, including diabetes, cancer, and aging. This study was designed to determine whether a variation in extracellular redox state would alter the function of insulin-resistant PC12 cells. Various redox states were established by providing different extracellular Cys/CySS Eh to insulin-resistant PC12 cells. We intensively investigated the relationship between redox state and catecholamine biosynthesis in PC12 cells, and evaluated the changes in cellular reactive oxygen species (ROS), catecholamine (CA) synthesis, tyrosine hydroxylase (TH) expressions, and the activity of rate-limiting enzyme in CA synthesis by using DCF-fluorescence, HPLC, and the real-time PCR, respectively. We also determined the protein levels of NF-E2-related factor 2 (Nrf2), a redox sensitive transcription factor, using an ELISA assay. We found that the oxidized Cys/CySS Eh (0 mV) pretreatment decreased CA, TH, and Nrf2 levels, but induced ROS overproduction. Insulin induced a significant increase in CA synthesis and ROS production, blocked by more reducing redox conditions. The paradox of CA and TH alterations between insulin and 0 mV groups may be attributed to degree of redox imbalance as evidenced by different ROS levels in 2 groups, which is further confirmed by CA alterations in different concentrations of hydrogen peroxide. Additionally, dithiole-3-thione (D3T, an inducer of Nrf2) corrected 0 mV-induced TH inhibition. In conclusion, CA biosynthesis in insulin-resistant PC12 cells could be influenced by extracellular Cys/CySS redox effects on cellular redox sensitive transcription factors.

The effect of a high vs. low glycemic response diet on the intra‐ and extracellular redox state (641.10)

Increased consumption of processed foods is suspected to be contributing to obesity and chronic disease. We hypothesized that long‐term consumption of more processed food (mimicked with high (HGR) or low glycemic response (LGR) starches) would lead to oxidation of the circulating redox state, increased fat accumulation, and reduced insulin sensitivity. Mice consumed HGR or LGR food for 16 weeks. Body mass increased at a faster rate for those consuming the HGR diet (p &lt; 0.05 for weeks 8‐16). Percentage body fat was greater and percentage lean mass was lower in the HGR group starting at 12 weeks (p &lt; 0.05; MRI). However, insulin sensitivity at week 14 (OGTT; AUC, p = 0.14) and metabolic parameters (RER, heat production, activity) at weeks 4 or 15 were not significantly different between groups. At the termination of the study, redox pairs (L/P, B/A) suggested a more oxidized redox state in blood of mice on the LGR diet. TrxR activity in blood and TBARS of blood and liver tissues were similar between groups (p &gt; 0.05). These data illustrate that the mechanism by which processed carbohydrates contributes to obesity is not via oxidation of the circulating redox state.Grant Funding Source: United States Department of Agriculture

Redox Imbalance and Biochemical Changes in Cancer

For this article, we explore a hypothesis involving the possible role of reduction/oxidation (redox) state in cancer. We hypothesize that many modifications in cellular macromolecules, observed in cancer progression, may be caused by redox imbalance. Recent biochemical data suggest that human prostate cancer cell lines show a redox imbalance (oxidizing) compared with benign primary prostate epithelial cells; the degree of oxidation varied with aggressive behavior of each cell line. Our recent data suggest that human breast cancer tissues show a redox imbalance (reducing) compared with benign adjacent breast tissues. Accumulating data summarized in this article suggest that redox imbalance may regulate gene expression and alter protein stability by posttranslational modifications, in turn modulating existing cellular programs. Despite significant improvements in cancer therapeutics, resistance occurs, and redox imbalance may play a role in this process. Studies show that some cancer therapeutic agents increase generation of reactive oxygen/nitrogen species and antioxidant enzymes, which may alter total antioxidant capacity, cause cellular adaptation, and result in reduced effectiveness of treatment modalities. Approaches involving modulations of intra- and extracellular redox states, in combination with other therapies, may lead to new treatment options, especially for patients who are resistant to standard treatments.

Evaluation of the total oxidant and antioxidant status of the plasma of dogs anaesthetised with isoflurane

Oxidative stress is an imbalance between free radical production and antioxidant defence. It causes the cellular and extracellular redox state to be shifted to the oxidative side by the disruption...

Abstract 5623: Extracellular thiols and system xc− play a significant role in non-targeted in vitro activity of a folate-tubulysin B conjugate.

Abstract Several folate-targeted small-molecule drug conjugates (SMDCs) are under development for the treatment of folate receptor-positive (FR+) cancer. To date, all clinical SMDCs have been constructed with a disulfide-based linker strategically placed in between the targeting ligand (e.g. folate) and the therapeutically active drug. At least for SMDCs, the disulfide linker remains relatively stable in circulation, but it rapidly breaks once the SMDC enters the reductive endocytic process within the targeted cell. While this structural design has proven to be successful for folate-based SMDCs constructed with mitomycins, maytansinoids, vinca alkaloids, and epothilones, the development of a more potent folate-tubulysin B conjugate (herein called EC0531) has revealed a non-FR-specific component related to in vitro cytotoxic activity, particularly at drug concentrations exceeding that required to saturate the FR. Rationalizing that the potential instability of the disulfide linker may contribute to this effect, we explored the impact of extracellular thiols on non-FR-specific activity of EC0531. Thus, when KB cells (FR+) and A549 cells (FR−) were co-treated with high-dose EC0531 (1 μM) in the presence of cell-impermeable thiol inhibitors, such as 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) or N-(2-carboxyethyl)maleimide (NCEM), non-FR-specific cytotoxicity was inhibited along with reduced non-specific uptake of 3H-EC0531 (tritium in the tubulysin moiety). We next found that independent of cell type or FR expression status, eliminating cystine and glutathione from the incubation medium significantly attenuated the non-specific activity of EC0531; this maneuver also significantly reduced the cellular efflux of extracellular thiols, such as cysteine, which we confirmed is the predominant thiol released by the cell, and at levels reaching 11-42 μM (for KB and A549 cells, respectively) following only a 2-hour conditioning period at 37°C. System xc− is a cystine/glutamate antiporter which has been shown to be involved in regulating the extracellular redox state of the cysteine/cystine couple in vitro. Thus, using system xc− inhibitors (e.g. sulfasalazine), we observed significant reductions in: (i) extracellular thiol activity; (ii) uptake of 3H-EC0531 (i.e. 3H-tubulysin); and (iii) non-specific activity of EC0531. Finally, siRNA-mediated knockdown of xCT (the functional subunit of system xc−) also caused dramatic reduction of extracellular thiol activity as well as reduced non-FR-mediated activity of EC0531. Taken together, our data support the involvement of extracellular thiols (mainly cysteine) in the extracellular reduction of disulfide-containing SMDCs to release the active drug payload which, in turn, may allow for non-specific drug uptake and possible activity, especially when the extracellular SMDC concentration is exceedingly high. Citation Format: Nikki Parker, Michael R. Pugh, Le-Cun Xu, Iontcho R. Vlahov, Christopher P. Leamon. Extracellular thiols and system xc− play a significant role in non-targeted in vitro activity of a folate-tubulysin B conjugate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5623. doi:10.1158/1538-7445.AM2013-5623

Peroxiredoxin 4: a multifunctional biomarker worthy of further exploration

Currently, there is much interest in identifying clinically relevant biomarkers, as they have the potential to be high utility non-invasive tools for early diagnosis and reliable patient monitoring in numerous conditions. Since its discovery almost 15 years ago, research on the ubiquitous antioxidant enzyme peroxiredoxin 4 (Prx4) has culminated in the recognition that Prx4 levels are different in blood drawn from the healthy general population and patients with acute or chronic diseases. In this commentary, the most striking research data from different in vitro approaches, animal models and human observational studies are discussed collectively, highlighting the clinical importance of Prx4 as a multifunctional staging and prognosis biomarker. In this context, the oxidative state of patients may be reflected by intra- and extracellular Prx4 levels, redox state, oligomerization and nitro-oxidative modifications of the enzyme. A consolidated model of the potential role and origin of circulating Prx4 is presented to stimulate further investigations in light of the current biomarker situation.

Regulation of prostate cancer cell invasion by modulation of extra- and intracellular redox balance

Recent metabolic profiles of human prostate cancer tissues showed a significant increase in cysteine (Cys) and a significant decrease in reduced glutathione (GSH) during cancer progression from low- to high-grade Gleason scores. Cys is primarily localized extracellularly, whereas GSH is present mostly inside the cell. We hypothesized that extra- or intracellular redox state alterations differentially regulate cell invasion in PC3 prostate carcinoma cells versus PrEC normal prostate epithelial cells. Cells were exposed to media with calculated Cys/CySS redox potentials ( E hCySS) ranging from − 60 to − 180 mV. After 3 h exposure to a reducing extracellular redox state ( E hCySS = − 180 mV), matrix metalloprotease (MMP), gelatinase, and NADPH oxidase activities increased, correlating with increases in cell invasion, cell migration, and extracellular hydrogen peroxide levels in PC3 cells but not PrECs. Knockdown of NADPH oxidase or MMP with silencing RNAs during cultivation with E hCySS = − 180 mV medium significantly decreased PC3 cell invasion. Modulation of extra- and intracellular redox states by exposure of PC3 cells to Cys/CySS-free medium (approx E hCySS = − 87 mV) containing 500 μM N-acetylcysteine resulted in a more reducing intracellular redox state and a significant decrease in cell invasive ability. The decrease in PC3 cell invasion induced by these conditions correlated with a decrease in MMP activity. Our studies demonstrated that an extracellular redox state that was more reducing than a physiologic microenvironment redox state increased PC3 cancer cell invasive ability, whereas an intracellular redox environmental that was more reducing than an intracellular physiologic redox state inhibited PC3 cell invasive ability.

HOW DOES ANESTHESIA AFFECT VARIOUS LEVELS OF EXPERIMENTAL TRAUMATIC BRAIN INJURY?

The use of anesthetics is a well-known treatment for severely injured patients. In the present study we tested the pathophysiology of several levels of injury damage in a rat model and also tested the effect of Equithesin on brain vitality in these models. Traumatic Brain Injury (TBI) was induced using the fluid percussion injury model in four levels: mild, moderate and two levels of severe TBI. Brain real-time evaluation was performed by the multiparametric monitoring assembly (MPA) which enable cerebral blood flow (CBF) monitoring by laser Doppler flowmetry, mitochondrial NADH (Nicotinamide adenine dinucleotide) monitoring by the fluorometric technique, ionic homehostasis using special mini-electrodes, intracranial pressure (ICP) by the ICP camino device and needle electrodes for ECoG (Electrocorticogram) recording. Our results showed high correlation between the level of impact and the extent of changes in the physiological properties of the injury as indicated by the changes in all parameters monitored using the MPA device. Moreover, Equithesin improved CBF, ionic extracellular level and mitochondrial redox state following mild and moderate TBI while in severe TBI, Equithesin did not improve the metabolic state of the cerebral cortex, although it decreased the mortality rate from 66% to 20%, and following extra-severe TBI level, Equithesin did not improve survival rate. In conclusion it seems that Equithesin's protective effect exists under mild to moderate levels of injury and not in case of severe injuries.

The Cystine/Cysteine Cycle and GSH Are Independent and Crucial Antioxidant Systems in Malignant Melanoma Cells and Represent Druggable Targets

Cancer chemoresistance is often due to upregulation of antioxidant systems. Therapeutic targeting of these systems is however hampered by their redundancy. Here, we have performed a functional dissection of the antioxidant systems in different melanoma cases aimed at the identification of the most effective redox active drug. We have identified two crucial antioxidant mechanisms: glutathione (GSH), the major intracellular redox buffer, and the cystine/cysteine cycle, which switches the extracellular redox state from an oxidized to a reduced state. The two mechanisms are independent in melanoma cells and may be substitutes for each other, but targeting both of them is lethal. Exposure to the pro-oxidant compound As(2)O(3) induces an antioxidant response. However, while in these cells the intracellular redox balance remains almost unaffected, a reduced environment is generated extracellularly. GSH depletion by buthioninesulfoximine (BSO), or cystine/cysteine cycle inhibition by (S)-4-carboxyphenylglycine (sCPG), enhanced the sensitivity to As(2)O(3). Remarkably, sCPG also prevented the remodeling of the microenvironment redox state. We propose that the definition of the prevalent antioxidant system(s) in tumors is crucial for the design of tailored therapies involving redox-directed drugs in association with pro-oxidant drugs. In melanoma cells, BSO is the best enhancer of As(2)O(3) sensitivity. However, since the strong remodeling of the microenvironmental redox state caused by As(2)O(3) may promote tumor progression, the concomitant use of cystine/cysteine cycle blockers is recommended.