Screening compound libraries for H2O2-mediated cancer therapeutics using a peroxiredoxin-based sensor.

Compounds that modulate H2O2 reaction networks have applications as targeted cancer therapeutics, as a subset of cancers exhibit sensitivity to this redox signal. Previous studies to identify therapeutics that induce oxidants have relied upon probes that respond to many different oxidants in cells, and thus do not report on only H2O2, a redox signal that selectively oxidizes proteins. Here we use a genetically encoded fluorescent probe for human peroxiredoxin-2 (Prx2) oxidation in screens for small-molecule compounds that modulate H2O2 pathways. We further characterize cellular responses to several compounds selected from the screen. Our results reveal that some, but not all, of the compounds enact H2O2-mediated toxicity in cells. Among them, SMER3, an antifungal, has not been reported as an oxidant-inducing drug. Several drugs, including cisplatin, that previously have been shown to induce reactive oxygen species (ROS) do not appear to oxidize Prx2, suggesting H2O2 is not among the ROS induced by those drugs.

A xenograft and cell line model of SDH-deficient pheochromocytoma derived from Sdhb+/- rats.

Tumors caused by loss-of-function mutations in genes encoding TCA cycle enzymes have been recently discovered and are now of great interest. Mutations in succinate dehydrogenase (SDH) subunits cause pheochromocytoma/paraganglioma (PCPG) and syndromically associated tumors, which differ phenotypically and clinically from more common SDH-intact tumors of the same types. Consequences of SDH deficiency include rewired metabolism, pseudohypoxic signaling and altered redox balance. PCPG with SDHB mutations are particularly aggressive, and development of treatments has been hampered by lack of valid experimental models. Attempts to develop mouse models have been unsuccessful. Using a new strategy, we developed a xenograft and cell line model of SDH-deficient pheochromocytoma from rats with a heterozygous germline Sdhb mutation. The genome, transcriptome and metabolome of this model, called RS0, closely resemble those of SDHB-mutated human PCPGs, making it the most valid model now available. Strategies employed to develop RS0 may be broadly applicable to other SDH-deficient tumors.

Monitoring the action of redox-directed cancer therapeutics using a human peroxiredoxin-2-based probe.

Redox cancer therapeutics target the increased reliance on intracellular antioxidant systems and enhanced susceptibility to oxidant-induced stress of some cancer cells compared to normal cells. Many of these therapeutics are thought to perturb intracellular levels of the oxidant hydrogen peroxide (H2O2), a signaling molecule that modulates a number of different processes in human cells. However, fluorescent probes for this species remain limited in their ability to detect the small perturbations induced during successful treatments. We report a fluorescent sensor based upon human peroxiredoxin-2, which acts as the natural indicator of small H2O2 fluctuations in human cells. The new probe reveals peroxide-induced oxidation in human cells below the detection limit of current probes, as well as peroxiredoxin-2 oxidation caused by two different redox cancer therapeutics in living cells. This capability will be useful in elucidating the mechanism of current redox-based therapeutics and in developing new ones.

A unique model for SDH-deficient GIST: an endocrine-related cancer.

We describe a unique patient-derived xenograft (PDX) and cell culture model of succinate dehydrogenase-deficient gastrointestinal stromal tumor (SDH-deficient GIST), a rare mesenchymal tumor that can occur in association with paragangliomas in hereditary and non-hereditary syndromes. This model is potentially important for what it might reveal specifically pertinent to this rare tumor type and, more broadly, to other types of SDH-deficient tumors. The primary tumor and xenografts show a very high proliferative fraction, and distinctive morphology characterized by tiny cells with marked autophagic activity. It is likely that these characteristics resulted from the combination of the germline SDHB mutation and a somatic KRAS G12D mutation. The most broadly relevant findings to date concern oxygen and oxidative stress. In paragangliomas harboring SDHx mutations, both hypoxic signaling and oxidative stress are putative drivers of tumor growth. However, there are no models for SDH-deficient paragangliomas. This related model is the first from a SDHB-mutated human tumor that can be experimentally manipulated to study mechanisms of oxygen effects and novel treatment strategies. Our data suggest that tumor growth and survival require a balance between protective effects of hypoxic signaling vs deleterious effects of oxidative stress. While reduced oxygen concentration promotes tumor cell survival, a further survival benefit is achieved with antioxidants. This suggests potential use of drugs that increase oxidative stress as novel therapies. In addition, autophagy, which has not been reported as a major finding in any type of SDH-deficient tumor, is a potential target of agents that might trigger autophagic cell death.

A mathematical analysis of Prx2-STAT3 disulfide exchange rate constants for a bimolecular reaction mechanism.

Appreciation of peroxiredoxins as the major regulators of H2O2 concentrations in human cells has led to a new understanding of redox signaling. In addition to their status as the primary reducers of H2O2 to water, the oxidized peroxiredoxin byproduct of this reaction has recently been shown capable of participation in H2O2-mediated signaling pathways through disulfide exchange reactions with the transcription factor STAT3. The dynamics of peroxidase-transcription factor disulfide exchange reactions have not yet been considered in detail with respect to how these reactions fit into the larger network of competing reactions in human cells. In this study, we used a kinetic model of oxidation and reduction reactions related to H2O2 metabolism in the cytosol of human cells to study the dynamics of peroxiredoxin-2 mediated oxidation of the redox-regulated transcription factor STAT3. In combination with previously reported experimental data, the model was used to estimate the rate coefficient of a biomolecular reaction between Prx2 and STAT3 for two sets of assumptions that constitute lower and upper bound cases. Using these estimates, we calculated the relative rates of the reaction of oxidized peroxiredoxin-2 and STAT3 and other competing reactions in the cytosol. These calculations revealed that peroxiredoxin-2-mediated oxidation of STAT3 likely occurs at a much slower rate than competing reactions in the cytosol. This analysis suggests the existence of more complex mechanisms, potentially involving currently unknown protein-protein recognition partners, which facilitate disulfide exchange reactions between peroxiredoxin-2 and STAT3.

Using Sensors and Generators of H2O2 to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate.

AIMS: Chemotherapeutics target vital functions that ensure survival of cancer cells, including their increased reliance on defense mechanisms against oxidative stress compared to normal cells. Many chemotherapeutics exploit this vulnerability to oxidative stress by elevating the levels of intracellular reactive oxygen species (ROS). A quantitative understanding of the oxidants generated and how they induce toxicity will be important for effective implementation and design of future chemotherapeutics. Molecular tools that facilitate measurement and manipulation of individual chemical species within the context of the larger intracellular redox network present a means to develop this understanding. In this work, we demonstrate the use of such tools to elucidate the roles of H2O2 and glutathione (GSH) in the toxicity mechanism of two ROS-based chemotherapeutics, piperlongumine and phenethyl isothiocyanate. RESULTS: Depletion of GSH as a result of treatment with these compounds is not an important part of the toxicity mechanisms of these drugs and does not lead to an increase in the intracellular H2O2 level. Measuring peroxiredoxin-2 (Prx-2) oxidation as evidence of increased H2O2, only piperlongumine treatment shows elevation and it is GSH independent. Using a combination of a sensor (HyPer) along with a generator (D-amino acid oxidase) to monitor and mimic the drug-induced H2O2 production, it is determined that H2O2 produced during piperlongumine treatment acts synergistically with the compound to cause enhanced cysteine oxidation and subsequent toxicity. The importance of H2O2 elevation in the mechanism of piperlongumine promotes a hypothesis of why certain cells, such as A549, are more resistant to the drug than others. INNOVATION AND CONCLUSION: The approach described herein sheds new light on the previously proposed mechanism of these two ROS-based chemotherapeutics and advocates for the use of both sensors and generators of specific oxidants to isolate their effects. Antioxid. Redox Signal. 24, 924-938.

A reaction-diffusion model of cytosolic hydrogen peroxide.

As a signaling molecule in mammalian cells, hydrogen peroxide (H2O2) determines the thiol/disulfide oxidation state of several key proteins in the cytosol. Localization is a key concept in redox signaling; the concentrations of signaling molecules within the cell are expected to vary in time and in space in manner that is essential for function. However, as a simplification, all theoretical studies of intracellular hydrogen peroxide and many experimental studies to date have treated the cytosol as a well-mixed compartment. In this work, we incorporate our previously reported reduced kinetic model of the network of reactions that metabolize hydrogen peroxide in the cytosol into a model that explicitly treats diffusion along with reaction. We modeled a bolus addition experiment, solved the model analytically, and used the resulting equations to quantify the spatiotemporal variations in intracellular H2O2 that result from this kind of perturbation to the extracellular H2O2 concentration. We predict that micromolar bolus additions of H2O2 to suspensions of HeLa cells (0.8 × 10(9)cells/l) result in increases in the intracellular concentration that are localized near the membrane. These findings challenge the assumption that intracellular concentrations of H2O2 are increased uniformly throughout the cell during bolus addition experiments and provide a theoretical basis for differing phenotypic responses of cells to intracellular versus extracellular perturbations to H2O2 levels.