Redox Modulation Research Articles

Platelet activation and ferroptosis mediated NETosis drives heme induced pulmonary thrombosis

Cell-free heme (CFH) is a product of hemoglobin, myoglobin and hemoprotein degradation, which is a hallmark of pathologies associated with extensive hemolysis and tissue damage. CHF and iron collectively induce cytokine storm, lung injury, respiratory distress and infection susceptibility in the lungs suggesting their key role in the progression of lung disease pathology. We have previously demonstrated that heme-mediated reactive oxygen species (ROS) induces platelet activation and ferroptosis. However, interaction of ferroptotic platelets and neutrophils, the mechanism of action and associated complications remain unclear. In this study, we demonstrate that heme-induced P-selectin expression and Phosphatidylserine (PS) externalization in platelets via ASK-1-inflammasome axis increases platelet-neutrophil aggregates in circulation, resulting in Neutrophil extracellular traps (NET) formation in vitro and in vivo. Further, heme-induced platelet activation in mice increased platelet-neutrophil aggregates and accumulation of NETs in the lungs causing pulmonary damage. Thus, connecting CFH-mediated platelet activation to NETosis and pulmonary thrombosis. As lung infections induce acute respiratory stress, thrombosis and NETosis, we propose that heme -mediated platelet activation and ferroptosis might be crucial in such clinical manifestations. Further, considering the ability of redox modulators and ferroptosis inhibitors like FS-1, Lpx-1 and DFO to inhibit heme-induced ferroptotic platelets-mediated NETosis and pulmonary thrombosis. They could be potential adjuvant therapy to regulate respiratory distress-associated clinical complications.

Mitochondria Need Their Sleep: Redox, Bioenergetics, and Temperature Regulation of Circadian Rhythms and the Role of Cysteine-Mediated Redox Signaling, Uncoupling Proteins, and Substrate Cycles.

Although circadian biorhythms of mitochondria and cells are highly conserved and crucial for the well-being of complex animals, there is a paucity of studies on the reciprocal interactions between oxidative stress, redox modifications, metabolism, thermoregulation, and other major oscillatory physiological processes. To address this limitation, we hypothesize that circadian/ultradian interaction of the redoxome, bioenergetics, and temperature signaling strongly determine the differential activities of the sleep-wake cycling of mammalians and birds. Posttranslational modifications of proteins by reversible cysteine oxoforms, S-glutathionylation and S-nitrosylation are shown to play a major role in regulating mitochondrial reactive oxygen species production, protein activity, respiration, and metabolomics. Nuclear DNA repair and cellular protein synthesis are maximized during the wake phase, whereas the redoxome is restored and mitochondrial remodeling is maximized during sleep. Hence, our analysis reveals that wakefulness is more protective and restorative to the nucleus (nucleorestorative), whereas sleep is more protective and restorative to mitochondria (mitorestorative). The "redox-bioenergetics-temperature and differential mitochondrial-nuclear regulatory hypothesis" adds to the understanding of mitochondrial respiratory uncoupling, substrate cycling control and hibernation. Similarly, this hypothesis explains how the oscillatory redox-bioenergetics-temperature-regulated sleep-wake states, when perturbed by mitochondrial interactome disturbances, influence the pathogenesis of aging, cancer, spaceflight health effects, sudden infant death syndrome, and diseases of the metabolism and nervous system.

Redox Modulation of Mitochondrial Proteins in the Neurotoxicant Models of Parkinson's Disease.

Significance: Mitochondrial proteins regulate the oxidative phosphorylation, cellular metabolism, and free radical generation. Redox modulation alters the mitochondrial proteins and instigates the damage to dopaminergic neurons. Toxicants contribute to Parkinson's disease (PD) pathogenesis in conjunction with aging and genetic factors. While oxidative modulation of a number of mitochondrial proteins is linked to xenobiotic exposure, little is known about its role in the toxicant-induced PD. Understanding the role of redox modulation of mitochondrial proteins in complex cellular events leading to neurodegeneration is highly relevant. Recent Advances: Many toxicants are shown to inhibit complex I or III and elicit free radical production that alters the redox status of mitochondrial proteins. Implication of redox modulation of the mitochondrial proteins makes them a target to comprehend the underlying mechanism of toxicant-induced PD. Critical Issues: Owing to multifactorial etiology, exploration of onset and progression and treatment outcomes needs a comprehensive approach. The article explains about a few mitochondrial proteins that undergo redox changes along with the promising strategies, which help to alleviate the toxicant-induced redox imbalance leading to neurodegeneration. Future Directions: Although mitochondrial proteins are linked to PD, their role in toxicant-induced parkinsonism is not yet completely known. Preservation of antioxidant defense machinery could alleviate the redox modulation of mitochondrial proteins. Targeted antioxidant delivery, use of metal chelators, and activation of nuclear factor erythroid 2-related factor 2, and combinational therapy that encounters multiple free radicals, could ameliorate the redox modulation of mitochondrial proteins and thereby PD progression.

Extracellular vesicles and redox modulation in aging

Extracellular vesicles and redox modulation in aging

Redox regulation of KV7 channels through EF3 hand of calmodulin.

Neuronal KV7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca2+-binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of KV7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves KV7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.

Intersubunit cross-linking is the key mechanism of cardiac ryanodine receptor dysfunction during oxidative stress: The role of cysteines 1078 and 2991.

The type 2 ryanodine receptor (RyR2) plays a key role in regulating cardiac intracellular Ca2+. We have previously shown that oxidative stress can regulate RyR2 by promoting the formation of disulfide bonds between neighboring RyR2 subunits (intersubunit cross-linking). However, the functional significance of this redox modification and cysteines involved in cross-linking remain unknown. Here, we used site-directed mutagenesis to replace cysteines 1078 and 2991 with serines in human RyR2 (hRyR2). Obtained constructs were expressed in HEK293 cells and treated with diamide to induce oxidative stress. Western blot analysis revealed that both mutations substantially protected against hRyR2 cross-linking. Analysis of the ER lumen [Ca2+] ([Ca2+]ER) dynamics with the ER Ca2+ sensor R-CEPIA1er in HEK293 cells expressing hRyR2 revealed that the mutation of either 1078 or 2991 cysteines prevented the depletion of ER Ca2+ load caused by RyR2 oxidation (by >40%). The simultaneous mutation of both cysteines produced the similar protection against hRyR2 cross-linking and [Ca2+]ER depletion, indicating a common mechanism of redox regulation involving both residues. Given the structural localization of both cysteines at the neighboring RyR2 subunits, we suggest that they form an intersubunit disulfide bond during channel oxidation, followed by the formation of the pathologically “leaky” channel. Although the N-terminal region (NTR) of RyR2 plays an important role in the channel's tetramer formation and functional regulation, the individual cysteines in this region are not directly involved in hRyR2 cross-linking. However, the structural integrity of the NTR has an allosteric effect on hRyR2 complex re-arrangements required for the channel's cross-linking. In conclusion, the results of this study revealed two critical cysteines within hRyR2 that are involved in the intersubunit disulfide cross-linking and the channel's functional response to oxidative stress.

Redox-Dependent Modulation of Human Liver Progenitor Cell Line Fate.

Redox homeostasis is determinant in the modulation of quiescence/self-renewal/differentiation of stem cell lines. The aim of this study consisted of defining the impact of redox modifications on cell fate in a human hepatic progenitor line. To achieve this, the HepaRG cell line, which shows oval ductular bipotent characteristics, was used. The impact of redox status on the balance between self-renewal and differentiation of HepaRG cells was investigated using different methodological approaches. A bioinformatic analysis initially proved that the trans-differentiation of HepaRG toward bipotent progenitors is associated with changes in redox metabolism. We then exposed confluent HepaRG (intermediate differentiation phase) to oxidized (H2O2) or reduced (N-acetylcysteine) extracellular environments, observing that oxidation promotes the acquisition of a mature HepaRG phenotype, while a reduced culture medium stimulates de-differentiation. These results were finally confirmed through pharmacological modulation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2), a principal modulator of the antioxidant response, in confluent HepaRG. NRF2 inhibition led to intracellular pro-oxidative status and HepaRG differentiation, while its activation was associated with low levels of reactive species and de-differentiation. In conclusion, this study shows that both intra- and extracellular redox balance are crucial in the determination of HepaRG fate. The impact of redox status in the differentiation potential of HepaRG cells is significant on the utilization of this cell line in pre-clinical studies.

Protein S-glutathionylation and sex dimorphic effects on hydrogen peroxide production by dihydroorotate dehydrogenase in liver mitochondria.

Dihydroorotate dehydrogenase (DHODH) oxidizes dihydroorotate to orotate for pyrimidine biosynthesis, donating electrons to the ubiquinone (UQ) pool of mitochondria. DHODH has a measurable rate for hydrogen peroxide (H2O2) production and thus contributes to cellular changes in redox tone. Protein S-glutathionylation serves as a negative feedback loop for the inhibition of H2O2 by several α-keto acid dehydrogenases and respiratory complexes in mitochondria, as well as ROS sources in liver cytoplasm. Here, we report this redox signaling mechanism also inhibits H2O2 production by DHODH in liver mitochondria isolated from male and female C57BL6N mice. We discovered that low amounts of the glutathionylation catalyst, disulfiram (50-500nM), almost abolished H2O2 production by DHODH in mitochondria from male mice. Similar results were collected with diamide, however, higher doses (1000-5000μM) were required to elicit this effect. Disulfiram and diamide also significantly suppressed H2O2 production by DHODH in female liver mitochondria. However, liver mitochondria from female mice were more resistant to disulfiram or diamide-mediated inhibition of H2O2 genesis when compared to samples from males. Analysis of the impact of disulfiram and diamide on DHODH activity revealed that both compounds inhibited the dehydrogenase directly, however the effect was less in female mice. Additionally, disulfiram and diamide impeded the use of dihydroorotate fueled oxidative phosphorylation in mitochondria from males and females, although samples collected from female rodents displayed more resistance to this inhibition. Taken together, our findings demonstrate H2O2 production by DHODH can be inhibited by glutathionylation and sex can impact this redox modification.

Influenza virus replication is affected by glutaredoxin1-mediated protein deglutathionylation.

Several redox modifications have been described during viral infection, including influenza virus infection, but little is known about glutathionylation and this respiratory virus. Glutathionylation is a reversible, post-translational modification, in which protein cysteine forms transient disulfides with glutathione (GSH), catalyzed by cellular oxidoreductases and in particular by glutaredoxin (Grx). We show here that (i) influenza virus infection induces protein glutathionylation, including that of viral proteins such as hemagglutinin (HA); (ii) Grx1-mediated deglutathionylation is important for the viral life cycle, as its inhibition, either with an inhibitor of its enzymatic activity or by siRNA, decreases viral replication. Overall these data contribute to the characterization of the complex picture of redox regulation of the influenza virus replication cycle and could help to identify new targets to control respiratory viral infection.

N-Acetyl Cysteine Effects on Radiation-induced Brain Injury in Rats: Redox, Inflammatory and Apoptotic Modulations

N-Acetyl Cysteine Effects on Radiation-induced Brain Injury in Rats: Redox, Inflammatory and Apoptotic Modulations

Succinylation and redox status in cancer cells.

Succinylation is a post-translational modification (PTM) event that associates metabolic reprogramming with various pathological disorders including cancers via transferring a succinyl group to a residue of the target protein in an enzymic or non-enzymic manner. With our incremental knowledge on the roles of PTM played in tumor initiation and progression, relatively little has been focused on succinylation and its clinical implications. By delineating the associations of succinylation with cancer hallmarks, we identify the, in general, promotive roles of succinylation in manifesting cancer hallmarks, and conceptualize two working modes of succinylation in driving oncogenic signaling, i.e., via altering the structure and charge of target proteins towards enhanced stability and activity. We also characterize succinylation as a reflection of cellular redox homeostatic status and metabolic state, and bring forth the possible use of hyper-succinylated genome for early cancer diagnosis or disease progression indication. In addition, we propose redox modulation tools such as cold atmospheric plasma as a promising intervention approach against tumor cells and cancer stemness via targeting the redox homeostatic environment cells established under a pathological condition such as hypoxia. Taken together, we emphasize the central role of succinylation in bridging the gap between cellular metabolism and redox status, and its clinical relevance as a mark for cancer diagnosis as well as a target in onco-therapeutics.

Dual-Site Chemosensor for Monitoring ·OH-Cysteine Redox in Cells and In Vivo

The reaction between hydroxyl radical (·OH) and cysteine (Cys) plays an important role in the redox balance of living cells. A deeper insight into this intracellular reaction modulation and process is necessary and draws great interest. A highly effective technique consists of the real-time visualization of the two bioactive species and the perception of their respective changes by using a fluorescent probe. In this study, a dual-site chemosensor SPI based on phenothiazine-cyanine was developed, which realized quantitative detection and real-time imaging of ·OH and Cys at their own fluorescence channels (·OH: λex = 485 nm, λem = 608 nm; Cys: λex = 426 nm, λem = 538 nm) without spectral crosstalk. The fluorescent sensor showed excellent anti-interference and selectivity for common biological substances, apart from the successful imaging of exogenous and endogenous ·OH and Cys. We further visualized the redox dynamic reaction and explored the correlation of ·OH and Cys generated by different inhibitors (sulfasalazine and (1S, 3R)-RSL3). Notably, the chemosensor also possesses the capacity to clearly monitor ·OH and Cys in living mice and zebrafish. This study reports on the first chemosensor to investigate the process of intracellular redox modulation and control between ·OH and Cys, which show potential to further explore some metabolic and physiological mechanisms.

Receptor-Mediated Redox Imbalance: An Emerging Clinical Avenue against Aggressive Cancers.

Cancer cells are more vulnerable to abnormal redox fluctuations due to their imbalanced antioxidant system, where cell surface receptors sense stress and trigger intracellular signal relay. As canonical targets of many targeted therapies, cell receptors sensitize the cells to specific drugs. On the other hand, cell target mutations are commonly associated with drug resistance. Thus, exploring effective therapeutics targeting diverse cell receptors may open new clinical avenues against aggressive cancers. This paper uses focused case studies to reveal the intrinsic relationship between the cell receptors of different categories and the primary cancer hallmarks that are associated with the responses to external or internal redox perturbations. Cold atmospheric plasma (CAP) is examined as a promising redox modulation medium and highly selective anti-cancer therapeutic modality featuring dynamically varying receptor targets and minimized drug resistance against aggressive cancers.

Targeting mitochondria for healthy brain aging: a case study of non‐psychoactive cannabinoids to rescue mitochondrial dysfunction in Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease (AD) arises from complex and multifaceted pathogenic mechanisms. Although many alterations occur in the aging brain that are likely to contribute to AD, there is growing evidence that mitochondrial inefficiency and dysfunction are one of the pathophysiological changes contributing to neurodegeneration in AD. The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway could be used to identify novel AD drug candidates that act by preserving mitochondrial function.MethodsAiming to identify AD drug candidates from natural products that preserve mitochondrial function, we have focused on phytochemical libraries with medicinal properties. We used a novel preclinical drug‐screening platform based on phenotypic assays that mimic different cellular toxicities and stresses associated with the AD brain that are characterized by mitochondrial dysfunction. To elucidate the molecular mechanisms for mitochondrial regulation, we harnessed an inducible, mitochondrially‐depleted neuronal cell line and employed the combined techniques of live‐cell imaging, super‐resolution microscopy, real‐time mitochondrial metabolic analysis, biochemical assays, immunoblotting, and qPCR.ResultsUsing this combined approach, we discovered that cannabinol (CBN), a non‐psychoactive cannabinoid, is a novel and potent neuroprotector. We demonstrated that not only does CBN protect neurons from oxytosis/ferroptosis in a manner that is dependent on mitochondria, it does so independently of cannabinoid CB1/CB2 receptors. Specifically, CBN alleviates intraneuronal amyloid toxicity, directly targets mitochondria, and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis in neuronal cells. These neuroprotective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP‐activated protein kinase (AMPK) signaling which help to maintain mitochondrial homeostasis.ConclusionsThis study highlights the potential of mitochondrially‐targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction for healthy brain aging, as well as opportunities for the discovery and development of future therapeutics for Alzheimer’s disease and other age‐associated neurological disorders.

Reappraisal of oxidized HMGB1 as a mediator and biomarker.

HMGB1 is a dual-function protein that acts as a chromatin-binding protein and as a danger-associated molecular pattern (DAMP) when released from activated immune cells or injured tissue. In much of the HMGB1 literature, immunomodulatory effects of extracellular HMGB1 are proposed to depend on its oxidation state. However, many of the foundational studies for this model have been retracted or flagged with expressions of concern. The literature on HMGB1 oxidation reveals a diversity of redox proteoforms of HMGB1 that are inconsistent with current models of redox modulation regulating HMGB1 secretion. A recent study of acetaminophen toxicity has identified previously unrecognized HMGB1 oxidized proteoforms. HMGB1 undergoes oxidative modifications that could serve as pathology-specific biomarkers and drug targets.

Energy substrate metabolism and oxidative stress in metabolic cardiomyopathy.

Metabolic cardiomyopathy is an emerging cause of heart failure in patients with obesity, insulin resistance, and diabetes. It is characterized by impaired myocardial metabolic flexibility, intramyocardial triglyceride accumulation, and lipotoxic damage in association with structural and functional alterations of the heart, unrelated to hypertension, coronary artery disease, and other cardiovascular diseases. Oxidative stress plays an important role in the development and progression of metabolic cardiomyopathy. Mitochondria are the most significant sources of reactive oxygen species (ROS) in cardiomyocytes. Disturbances in myocardial substrate metabolism induce mitochondrial adaptation and dysfunction, manifested as a mismatch between mitochondrial fatty acid oxidation and the electron transport chain (ETC) activity, which facilitates ROS production within the ETC components. In addition, non-ETC sources of mitochondrial ROS, such as β-oxidation of fatty acids, may also produce a considerable quantity of ROS in metabolic cardiomyopathy. Augmented ROS production in cardiomyocytes can induce a variety of effects, including the programming of myocardial energy substrate metabolism, modulation of metabolic inflammation, redox modification of ion channels and transporters, and cardiomyocyte apoptosis, ultimately leading to the structural and functional alterations of the heart. Based on the above mechanistic views, the present review summarizes the current understanding of the mechanisms underlying metabolic cardiomyopathy, focusing on the role of oxidative stress.

Hydrogen sulfide and nitric oxide regulate the adaptation to iron deficiency through affecting Fe homeostasis and thiol redox modification in Glycine max seedlings

Iron (Fe) is a vital microelement required for the growth and development of plants. Hydrogen sulfide (H2S) and nitric oxide (NO), as messenger molecules, participated in the regulation of plant physiological processes. Here, we studied the interaction effects of H2S and NO on the adaptation to Fe deficiency in Glycine max L. Physiological, biochemical and molecular approaches were conducted to analyze the role of H2S and NO in regulating the adaptation to Fe deficiency in soybean. We found that H2S and NO had obvious rescuing function on the Fe deficiency-induced the plant growth inhibition, which was significantly correlated with the increase in Fe content in the leaves, stems, and roots of soybean. Meanwhile, H+-flux, ferric chelate reductase (FCR) activity, and root apoplast Fe content were significantly affected by H2S and NO. Under Fe deficiency conditions NO and H2S regulated the expression of genes related to Fe homeostasis. Moreover, photosynthesis (Pn) and photosystem II (PSII) efficiency were enhanced by H2S and NO, and thiol redox modification was important for regulating the adaptation of Fe deficiency. The aforementioned affirmative influences caused by H2S and NO were also totally reversed by cPTIO (a NO scavenger). Our results suggested that H2S might act upstream of NO in response to Fe deficiency by affecting the Fe homeostasis enzyme activities and gene expression, and by promoting Fe accumulation in plant tissues as well as by enhancing thiol redox modification and photosynthesis in soybean plants.

Wound Healing versus Metastasis: Role of Oxidative Stress.

Many signaling pathways, molecular and cellular actors which are critical for wound healing have been implicated in cancer metastasis. These two conditions are a complex succession of cellular biological events and accurate regulation of these events is essential. Apart from inflammation, macrophages-released ROS arise as major regulators of these processes. But, whatever the pathology concerned, oxidative stress is a complicated phenomenon to control and requires a finely tuned balance over the different stages and responding cells. This review provides an overview of the pivotal role of oxidative stress in both wound healing and metastasis, encompassing the contribution of macrophages. Indeed, macrophages are major ROS producers but also appear as their targets since ROS interfere with their differentiation and function. Elucidating ROS functions in wound healing and metastatic spread may allow the development of innovative therapeutic strategies involving redox modulators.

Redox Modulation by Tryptophan Metabolism: Role of Kynurenine-derived Electrophiles

Redox Modulation by Tryptophan Metabolism: Role of Kynurenine-derived Electrophiles

Early Monitoring of Redox Status Based on Reduced Molecules Using In Vivo DNP-MRI for Cancer Treatment

<h3>Purpose/Objective(s)</h3> Radiation treatment effectiveness is assessed through the observation of morphological changes with CT or MRI images after treatment. The treatment efficacy of radiotherapy depends on redox modulation in tumor tissue, such as hydroxyl radicals and of reactive oxygen species generated by irradiation. Therefore, redox status may be a prognostic factor response to radiotherapy. However, spatiotemporal response of the redox metabolism in tumor tissue post-irradiation remains unknown. The purpose of this study was to detect early changes in the redox status in tumor tissue after irradiation using <i>in vivo</i> dynamic nuclear polarization (DNP)-MRI, to clarify the key molecules that cause these changes, and to compare these changes with early changes in lactate metabolism using hyperpolarized ¹³C magnetic resonance spectroscopy (MRS). <h3>Materials/Methods</h3> To monitor the response of tumor redox status after irradiation, redox imaging using in vivo DNP MRI/ Carbamoyl PROXYL (CmP) as redox sensitive DNP probe was performed after 5 Gy irradiation. To clarify the relationship between redox status alteration and treatment efficacy, in vivo DNP MRI was performed before and after irradiation by 2 and 5 Gy. In addition, tissue GSH and AsA levels on day 1 post irradiation were determined by HPLC. To observe the Warburg effect after radiation treatment, dissolution DNP study using hyperpolarized ¹³C pyruvate was performed before and after irradiation. <h3>Results</h3> The DNP image intensity in the tumor demonstrated a maximum value approximately four minutes after CmP administration, thereafter the image intensity was decreased by the redox reaction. The tumor GSH and AsA level on day 1 post irradiation were decreased compared to the control group. Production of hyperpolarized ¹³C lactate in tumor tissue did not change before and after irradiation. <h3>Conclusion</h3> Our study highlights that redox status in tumor tissue is already significantly decreased on day 1 before the detection of any morphological changes, even after irradiation with a dose as low as 2 Gy. Early changes in this redox status may be related decreases in glutathione and ascorbic acid levels in the tumor tissue after irradiation, which may alter earlier than ¹³C pyruvate hyperpolarized MRS, based on lactate metabolism. Therefore, early assessment of change in decay rate using in vivo DNP-MRI/CmP probing may predict the subsequent radiotherapy effect.