The iron-chelating drug triapine causes pronounced mitochondrial thiol redox stress
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.Toxicology Letters (Impact Factor: 3.26). 03/2011; 201(2):130-6. DOI: 10.1016/j.toxlet.2010.12.017
Triapine (Tp) is an iron chelator with activity against several types of cancer. Iron-Tp [Fe(III)(Tp)(2)] can be redox-cycled to generate reactive oxygen species that may contribute to its cytotoxicity. However, evidence for this mechanism in cells is limited. The cytosolic and mitochondrial thioredoxins (Trx1 and Trx2, respectively) are essential for cell survival. They are normally maintained in the reduced state, and support the function of many intracellular proteins including the peroxiredoxins (Prxs). Their redox status can indicate oxidant stress in their respective subcellular compartments. Tp treatment of human lung A549 cells caused almost complete oxidation of Trx2 and its dependent peroxiredoxin (Prx3), but there was no effect on Trx1 redox status. Significant inhibition of total TrxR activity did not occur until Tp levels were 4-fold above those needed to cause Trx2 oxidation. While Tp caused a 36-45% decline in reduced glutathione (GSH) levels, GSH accounted for >99% of the total glutathione in the absence and presence of Tp. In vitro studies demonstrated that cysteine reduces Fe(III)(Tp)(2) to Fe(II)(Tp)(2), and cysteine was faster and more efficient than reduced glutathione (GSH) in this regard. Fe(III)(Tp)(2) also mediated the oxidation of purified Trx2 in vitro. Thus, Fe(III)(Tp)(2) itself, and/or various reactive species that may result from its redox cycling, could account for Trx2 and Prx3 oxidation in Tp-treated cells. The striking difference between the effects on Trx2 and Trx1 implies a pronounced thiol redox stress that is largely directed at the mitochondria. These previously unrecognized effects of Tp could contribute to its overall cytotoxicity.
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- "The results with multiple cell lines show that mitochondrial Prx3 is distinctly sensitive to the pro-oxidant effects of low micromolar Tp and sub-micromolar Dp44mT, with little to no effect on cytosolic Prx1 (Fig. 3, and Tp does not cause TrxR2 inhibition in cells. While nitric oxide is a potential Prx oxidant, the inability of the nitric oxide synthase inhibitor L-NAME to protect Prx3 from Tp (Fig. 10) suggests that nitric oxide does not have a major role. "
ABSTRACT: Peroxiredoxin-3 (Prx3) accounts for about 90% of mitochondrial peroxidase activity, and its marked upregulation in many cancers is important for cell survival. Prx3 oxidation can critically alter peroxide signaling and defense and can be a seminal event in promoting cell death. Here it is shown that this mechanism can be exploited pharmacologically by combinations of clinically available drugs that compromise Prx3 function in different ways. Clinically relevant levels of the thiosemicarbazone iron chelators triapine (Tp) and 2,2'-Dipyridyl-N,N-dimethylsemicarbazone (Dp44mT) promote selective oxidation of mitochondrial Prx3, but not cytosolic Prx1, in multiple human lung and ovarian cancer lines. Decreased cell survival closely correlates with Prx3 oxidation. However, Prx3 oxidation is not merely an indicator of cell death as cytotoxic concentrations of cisplatin do not cause Prx3 oxidation. The siRNA-mediated suppression of either Prx3 or thioredoxin-2, which supports Prx3, enhances Tp's cytotoxicity. Tp-mediated Prx3 oxidation is driven by enhanced peroxide generation, but not by nitric oxide. Many tumors overexpress thioredoxin reductase (TrxR) which supports Prx activity. Direct inhibitors of TrxR (e.g. auranofin, cisplatin) markedly enhanced Tp's cytotoxicity, and auranofin enhanced Prx3 oxidation by low dose Tp. Together, these results support an important role for Prx3 oxidation in the cytotoxicity of Tp, and demonstrate that TrxR inhibitors can significantly enhance Tp's cytotoxicity. Thiosemicarbazone-based regimens could prove effective for targeting Prx3 in a variety of cancers.
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ABSTRACT: Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper(II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)-Triapine are reduced to the iron(II)-Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron(II)-Triapine complex are formed. Formation of the iron(II)-Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical.
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ABSTRACT: The reactive aldehyde acrolein is a ubiquitous environmental pollutant and is also generated endogenously. It is a strong electrophile and reacts rapidly with nucleophiles including thiolates. This review focuses on the effects of acrolein on thioredoxin reductase (TrxR) and thioredoxin (Trx), which are major regulators of intracellular protein thiol redox balance. Acrolein causes irreversible effects on TrxR and Trx, which are consistent with the formation of covalent adducts to selenocysteine and cysteine residues that are key to their activity. TrxR and Trx are more sensitive than some other redox-sensitive proteins, and their prolonged inhibition could disrupt a number of redox-sensitive functions in cells. Among these effects are the oxidation of peroxiredoxins and the activation of apoptosis signal regulating kinase (ASK1). ASK1 promotes MAP kinase activation, and p38 activation contributes to apoptosis and a number of other acrolein-induced stress responses. Overall, the disruption of the TrxR/Trx system by acrolein could be significant early and prolonged events that affect many aspects of redox-sensitive signaling and oxidant stress.
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