Using Sensors and Generators of H[subscipt 2]O[subscript 2] to Elucidate the Toxicity Mechanism of Piperlongumine and Phenethyl Isothiocyanate

Abstract

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 H[subscript 2]O[subscript 2] 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 H[subscript 2]O[subscript 2] level. Measuring peroxiredoxin-2 (Prx-2) oxidation as evidence of increased H[subscript 2]O[subscript 2], 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 H[subscript 2O[subscript 2] production, it is determined that H[subscript 2]O[subscript 2] produced during piperlongumine treatment acts synergistically with the compound to cause enhanced cysteine oxidation and subsequent toxicity. The importance of H[subscript 2]O[subscript 2] 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.