Browsing by Subject "Reactive oxygen species"

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Engineering and validation of a human cyst(e)ine degrading enzyme as a novel cancer therapeutic
(2017-05) Cramer, Shira Lyla; Georgiou, George; Stone, Everett; Alper, Hal; Maynard, Jennifer; Ehrlich, Lauren; DiGiovanni, John
Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. As a precursor for the biosynthesis of GSH, L-cysteine (L-Cys) availability is critical for maintaining the intracellular thiol redox potential and under conditions of elevated ROS, endogenous L-Cys production is insufficient for GSH synthesis. This necessitates the import of extracellular L-cyst(e)ine (predominantly in its disulfide form, L-cystine (CSSC)) to meet cellular antioxidant requirements. Since L-Cys is a non-essential amino acid in animals, eliminating L-Cys and CSSC uptake should selectively impact tumors that display increased ROS production and thus exhibit a higher demand for antioxidants, without causing an adverse effect on normal physiology. This can be accomplished by eliminating the extracellular pool of L-Cys and CSSC through the action of an enzyme that selectively converts these amino acids into non-toxic products. Unfortunately, no human enzyme displays sufficient catalytic properties towards both L-Cys and CSSC to be relevant for clinical applications. In the chapters that follow, we describe the engineering of a human L-Cys and CSSC degrading enzyme (cyst(e)inase) as a novel and potent therapeutic for tumors displaying elevated levels of ROS. We show that administration of cyst(e)inase mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates at a therapeutically useful rate. In a wide variety of models, treatment with this enzyme suppresses tumor growth in mice, yet results in no apparent toxicities even after months of continuous treatment. Through additional engineering, we describe the isolation of novel enzymes that display the requisites for clinical development; including increases in soluble protein yields and catalytic activity towards both L-Cys and CSSC, which together translates to practical doses for a human therapeutic at reasonable manufacturing costs. Finally, upon further investigation into the therapeutic effect of cyst(e)inase, we show that cyst(e)inase treatment dramatically inhibits metastasis, as well as suggesting an important role of ROS regulation and the immune system in a syngeneic mouse model.
Exercise training reduces reactive oxygen species production in myocardial mitochondria of rat hearts
(2005-05-21) Barnes, Brian Douglas; Starnes, Joseph W.
Mitochondria are recognized to be a major cellular site of reactive oxygen species (ROS) production which is elevated during exercise. Exercise is also known to provide cardioprotection against ischemia/reperfusion injury following Ca²⁺ accumulation, but the effect of exercise on mitochondrial ROS production following Ca²⁺ remains unclear. The purpose of this study was to test the hypthesis that exercise results in a decrease of mitochondrial ROS in the heart. Adult male, 9 month old, Fisher 344 rats were randomly assigned to one of two treatment groups: sedentary control (SED) (n = 8) or exercised trained (ET) (n = 11). Trained rats ran on a motorized treadmill for 10 weeks (5- days/week, 60 min/day, 25m/min, and 6° grade). Heart mitochondria were isolated and oxidative phosphorylation measures determined to assure that high quality preparations were used. H₂O₂ production, an indicator of ROS production, was detected by fluorescence (Amplex Red) using succinate as substrate. Mitochondrial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) was also determined. No differences were observed between SED and ET for oxidative Phosphorylation parameters including State 3 rate, respiratory control ratio (State 3/State 4), and ADP:O ratio. Likewise, mitochondrial SOD and GPx activities were not affected by exercise. CAT activity was very low compared to SOD and GPx, but its activity was increased by 49% (from 0.59±0.03 to 0.88± 0.11 Units/mg protein, P<0.05) after exercise training. H₂O₂ production by ET was lower than SED in the absence of calcium (323±12 vs 362±11 pmol H₂O₂ /min/mg protein, P<0.05) and in the presence of 50 [Micrometer] Ca²⁺ (154±3 vs 197±7, P<0.05). Addition of ROT, an inhibitor of complex I in the electron transport chain (ETC), greatly reduced H₂O₂ production and eliminated differences between ET and SED in both the absence and presence of calcium. We conclude that exercise training greatly reduces ROS production in myocardial mitochondria primarily through adaptations specific to complex I
Human enzyme depletion of cyst(e)ine for pancreatic cancer therapy
(2018-05-02) Kshattry, Sabin; DiGiovanni, John; Georgiou, George; Stone, Everett; Tiziani, Stefano
Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate at 8%. Since targeting KRAS, the driver oncogene in PDAC, has not been a clinically successful endeavor to date and given that the growth of pancreatic tumors is intricately linked to reactive oxygen species (ROS) detoxification mechanisms, perturbation of oxidative balance might be an effective therapeutic strategy. A major intracellular antioxidant is glutathione (GSH), a tripeptide made of glycine, glutamate and cysteine. Cysteine (L-Cys), which has the functional moiety of GSH, can either be synthesized de novo intracellularly or imported, predominantly as cystine (CSSC) that is reduced intracellularly to L-Cys. Recently, our group has engineered a human enzyme called cyst(e)inase that degrades both extracellular L-Cys and CSSC and is a safe and effective therapeutic agent for multiple cancer models. In our study with three pancreatic cancer cell lines, we found that even though cyst(e)inase depleted intracellular L-Cys and GSH in all three, only one “sensitive” cell line exhibited ROS accumulation and apoptotic cell death; the other two “resistant” cell lines were able to arrest their growth and maintain survival. Further mechanistic exploration showed that only the resistant cell lines were capable of maintaining mitochondrial fitness during L-Cys/CSSC depletion. Since resistance seemed predicated on maintenance of functional mitochondria, a major ROS producer, we sought to sensitize the resistant cells to cyst(e)inase by concurrently inhibiting other antioxidant pathways. As expected, this approach led to synergistic inhibition of cell survival in all three cell lines with the most striking effect and high translational potential provided by auranofin, a thioredoxin reductase inhibitor and an approved drug for rheumatoid arthritis. Treatment of cyst(e)inase and auranofin in combination caused a synergistic increase in mitochondrial ROS and apoptosis, and also led to inhibition of mitophagy – a cellular mechanism for mitochondrial quality control. Treatment of nude mice harboring pancreatic cancer xenografts recapitulated these results: auranofin sensitized the completely resistant cell line to cyst(e)inase without causing any systemic toxicity. Our data provides strong rationale to investigate the efficacy of cyst(e)inase and auranofin combination to treat patients with PDAC
Leveraging selective peptoid degradation for biosensing applications
(2022-08-12) McKenzie, Hattie Christine (Schunk); Rosales, Adrianne M.; Suggs, Laura J.
Development of multi-functional materials and biosensors that can achieve an in-situ response designed by the user is a current need in the biomaterials field, especially in complex biological environments, such as inflammation, where multiple enzymatic and oxidative signals are present. In the past decade, there has been extensive research and development of materials chemistries for detecting and monitoring enzymatic activity, as well as for releasing therapeutic and diagnostic agents in regions undergoing oxidative stress. However, there has been limited development of materials in the context of enzymatic and oxidative triggers together, despite their closely tied and overlapping mechanisms. One major fundamental design challenge to integrating multiple sensing elements in tandem is instability and uncontrolled cross-reactivity. Thus, to successfully detect biomarkers in synergy, there is need for innovative strategies in controlling biostability while maintaining well-defined bioactivity. We aim to address this challenge using synthetic, sequence-defined peptoids. Due to their N-substitution, peptoids are generally regarded as resistant to biological degradation, such as enzymatic and hydrolytic mechanisms. This stability is an especially attractive feature for therapeutic development and is a selling point of many previous biological studies. However, oxidative degradation of peptoids mediated by reactive oxygen and nitrogen species (ROS/RNS) is key mode of degradation that remains to be fully explored. ROS and RNS are biologically relevant in numerous contexts where biomaterials may be present, thus, improving understanding of peptoid oxidative susceptibility is crucial to exploit their full potential in the biomaterials field. Toward this end, we demonstrate a fundamental characterization of sequence-defined peptoid chains in the presence of chemically generated ROS, as compared to ROS-susceptible peptides such as proline and lysine oligomers. These results expand understanding of peptoid degradation to oxidative and enzymatic mechanisms, and demonstrate the potential for peptoid incorporation into materials where selectivity towards oxidative degradation is necessary, or directed enzymatic susceptibility is desired. By considering the materials chemistry of enzymatically and oxidatively triggered biomaterials in tandem, we hope to encourage synthesis of new biosensors that capitalize on their synergistic roles and overlapping mechanisms in inflammatory environments for future applications in disease diagnosis and monitoring.
NGFI-B redox sensitivity and regulation of mitochondrial bioenergetics
(2011-08) Abramson, Ellen M.; Mills, Edward Michael; Bratton, Shawn; Wright, Casey; Kline, Kimberly; Nunez, Nomeli
Changes in intracellular redox homeostasis are implicated in both normal cell signaling and as pathophysiological mechanisms contributing to a variety of age-related diseases, including diabetes, atherosclerosis, neurodegenerative conditions, and cancer. Though a variety of well described mechanisms exist to counterbalance the overproduction of cellular oxidants and maintain optimal intracellular redox poise, the understanding of the mechanism(s) through which cellular redox homeostasis regulates cell signaling functions is less well understood. Here, we demonstrate that signaling by the immediate early gene / orphan nuclear hormone receptor NGFI-B (Nur77, TR3), which functions pleiotropically in the regulation of cell growth, metabolism, differentiation and death in diverse tissues, is redox-regulated at both the level of induction and NGFI-B-dependent gene transcription. Using co-immunoprecipitation experiments in cells, we also identified a novel interaction between NGFI-B and the cytoplasmic thiol-reducing catalyst thioredoxin1 (Trx1), that, similar to DTT, blocks NGFI-B-dependent gene expression in a manner that depends on the Trx1 active site cysteines. Together these observations add NGFI-B-dependent gene expression to a growing portfolio of transcription factor pathways that are redox-regulated. NGFI-B, in addition, appears to regulate the mitochondrial membrane potential in L6 skeletal myoblasts. NGFI-B is indispensible for T-cell receptor-mediated apoptosis and induces cell death in a variety of cell types in response to diverse pro-apoptotic stimuli. Like p53, translocation of NGFI-B from the nucleus to the mitochondria may be a critical aspect of its pro-apoptotic function. Interestingly, we found that enforced NGFI-B expression in L6 skeletal muscle myoblasts led to a significant decrease of MMP that peaked 48hr after transfection and did not require a cell death-inducing stimulus. Moreover, NGFI-B transfected cells had no increase in mitochondrial cytochrome C release despite loss of MMP at 48 hr. Combined, these data suggest that loss of MMP in muscle cells may be an early event in the apoptotic process regulated by NGFI-B. This, along with the redox regulation of NGFI-B, provides unique evidence of a relationship between the mitochondria, mitochondrial by-products, ROS, and the regulation of and by the transcription factor NGFI-B.
Role of reactive oxygen species in pluripotent stem cells cardiac differentiation and survival
(2018-08) Tu, Chengyi; Zoldan, Janeta; Suggs, Laura; Baker, Aaron; Cooney, Austin
Pluripotent stem cells (PSCs) derived cardiomyocytes provide an invaluable cell source for numerous important applications. PSC-cardiomyocytes may be transplanted into injured hearts to repair the tissue damage caused by myocardial infarction. Patients-specific induced PSCs (iPSCs) derived cardiomyocytes can serve as platforms for personalized cardiotoxicity test and drug screening. Further, in vitro cardiac differentiation process may be employed as a model for studying heart development. To achieve efficient and consistent cardiac differentiation, a wide range of biochemical (e.g., growth factors and small molecules) and physical stimuli (e.g., mechanical stress and electrical stimulation) have been explored with various degrees of success. Reactive oxygen species (ROS) are known to be critical in cell signaling, mainly via their modifications of protein activities. ROS have been shown to be indispensable for cardiac differentiation. In this dissertation, we seek to utilize ROS as a potential new tool in the control of cardiac differentiation, maturation and removal of undifferentiated cells. Specifically, we modulated ROS by changing glucose level and/or applying antioxidants. Our results showed that differentiation of mouse embryonic stem cells (mESCs) in high glucose medium without any antioxidants resulted in high level of cellular ROS, and also increased cardiac differentiation efficiency from about 15% to over 40%, along with increased expression of cardiac markers such as NKX2.5 and MESP1. More interestingly, when mESCs were differentiated in the presence of commonly used thiol-containing antioxidants such as beta-mercaptoethanol (BME) or monothiol glycerol (MTG), the resulting cardiomyocytes showed lower TNNI3/TNNI1 expression ratio, lower beating frequency and slower contraction velocity, suggesting delayed cardiac maturation. In contrast, Trolox, a vitamin E derivative and a non-thiol antioxidant, did not have these effects despite its strong antioxidant efficacy. These results suggest that redox regulation of cardiac differentiation is not only dictated by overall cellular ROS, but fine-controlled on a subcellular level. Further, ROS may also be utilized to promote desired cellular apoptosis. When co-treated with GSK3 inhibitors and antioxidants such as N-acetyl cysteine (NAC), human iPSCs underwent rapid and extensive apoptosis. This unique effect might be leveraged to remove iPSCs from their differentiated descendants to minimize the future risk of tumorigenesis.