Using Sensors and Generators of H₂O₂to 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 2 O 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 2 O 2 level. Measuring peroxiredoxin-2 (Prx-2) oxidation as evidence of increased H 2 O 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 2 O 2 production, it is determined that H 2 O 2 produced during piperlongumine treatment acts synergistically with the compound to cause enhanced cysteine oxidation and subsequent toxicity. The importance of H 2 O 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.

Pseudomonas Quinolone Signal molecule PQS behaves like a B Class inhibitor at the I Q site of mitochondrial complex I

“…The observation that inhibition of complex I by Rotenone did not lead to an increase in mitochondrial ROS in intact A549 cells (unlike HeLa cells) confirms earlier observations that A549 cells are less sensitive to ROS‐inducing drug treatment . In contrast, isolated mitochondria from A549 cells also showed a ROS increase due to Rotenone (but not PQS) treatment, confirming the frequent observation that the cellular ROS defense mechanism may be lost during mitochondrial isolation . As described earlier, the effect of PQS on cells is concentration dependent, very high PQS values (100 µmol/L) led to cell death as indicated by the penetration of DAPI into cells, and significantly increased cellular and nuclear ROS values.…”

“…Future experiments must show whether the PQS effect on complex III is actually due only to the effect of a class B inhibitor or is also related to the iron‐chelating effect attributed to PQS . The observation that inhibition of complex I by Rotenone did not lead to an increase in mitochondrial ROS in intact A549 cells (unlike HeLa cells) confirms earlier observations that A549 cells are less sensitive to ROS‐inducing drug treatment . In contrast, isolated mitochondria from A549 cells also showed a ROS increase due to Rotenone (but not PQS) treatment, confirming the frequent observation that the cellular ROS defense mechanism may be lost during mitochondrial isolation .…”

“…44 In contrast, isolated mitochondria from A549 cells also showed a ROS increase due to Rotenone (but not PQS) treatment, confirming the frequent observation that the cellular ROS defense mechanism may be lost during mitochondrial isolation. 44 As described earlier, 29 the effect of PQS on cells is concentration dependent, very high PQS values (100 µmol/L) led to cell death as indicated by the penetration of DAPI into cells, and significantly increased cellular and nuclear ROS values. In summary, we conclude that PQS at concentrations as in the sputum range of CF patients is sufficient to inhibit complex I at the I Q site without stimulating ROS formation.…”

“…In HeLa cells, the effect of Rotenone was different: Rotenone significantly increased superoxide levels. It has to be mentioned that the effect of Rotenone on ROS production in cells is not consistent, depending on the ROS defense, which is obviously specific in A594 cells . To investigate whether Rotenone would in principle increase ROS in A594 cells, we additionally performed inhibitor treatment with isolated mitochondria from A549 cells based on observations that the ROS defense mechanism is often lost during mitochondrial isolation .…”

Mitochondrial H2O2 Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death

“…Efforts exist to both target existing redox-active compounds to the mitochondria and also understand the mechanisms of compounds that seem to act via the mitochondria 34,35 . Making controlled perturbations with a peroxide generator has been useful in elucidating drug mechanisms in the cytoplasm, and could be a key tool in understanding chemotherapeutics in the mitochondria 36 .…”