The iron complex of Dp44mT is redox-active and induces hydroxyl radical formation: An EPR study
Graphical Abstract
Dp44mT is a redox-active iron chelator developed for cancer chemotherapy. Recent studies have questioned the ability of Dp44mT to induce hydroxyl radical formation upon complexation with iron. We show this is an erroneous view and further discuss the relevance of redox activity to the biological effects of this molecule.
Introduction
Initially, iron chelation therapy was designed to lessen the toxic effects of excess iron evident in iron-overload diseases [1]. In these conditions, iron chelators are used to bind excess iron and prevent its ability to participate in Fenton chemistry and inhibit the formation of reactive oxygen species (ROS), such as the hydroxyl radical (OH) [2]. As such, iron chelators are ideal candidates for the treatment of iron-loading conditions e.g., β-thalassemia [3], [4].
Through specific ligand design initiatives [5], [6], the toxicological properties of some iron chelator complexes have shifted their intended application to cancer chemotherapy [7], [8]. By depleting iron from rapidly proliferating cancer cells, iron chelators can inhibit essential cellular processes such as DNA synthesis [6]. This is due, in part, to their ability to inhibit ribonucleotide reductase activity, which is the iron-containing enzyme involved in the rate-limiting step of DNA synthesis [9], [10], [11], [12]. In addition, cellular iron-depletion is known to affect the expression of molecules involved in metastasis and cell cycle progression, for example, N-myc downstream regulated gene 1, cyclin D1, p21 etc., leading to G1/S arrest [13], [14], [15]. Furthermore, some iron chelators, particularly those with soft electron donors, such as nitrogen and sulfur, are capable of enhancing the production of ROS after complexation with iron [6]. In contrast, predominately hard oxygen electron donors that form iron complexes with little or no redox activity are used for iron-overload disease [3], [6], [16].
We have designed the di-2-pyridylketone thiosemicarbazone (DpT) group of chelators specifically for the treatment of cancer and these were prepared based upon a series of structure-activity relationship studies in our laboratories [17], [18], [19]. The DpT chelators were designed to incorporate the thiosemicarbazone moiety, which was observed to confer high anti-proliferative activity against a number of tumor cell lines [20], [21], [22]. Of these ligands, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) (Fig. 1A) was particularly active and was a lead compound. Dp44mT is a tridentate ligand utilizing the pyridyl nitrogen, imine nitrogen and sulfur as donor atoms (N,N,S system; Fig. 1B), which complete the coordination shell of iron by binding in a 2:1 chelator to iron ratio [21]. In vivo studies using a mouse tumor model (M109) and a variety of human tumor xenografts in nude mice, confirmed the anti-neoplastic activity of Dp44mT [20], [23]. Importantly, no systemic iron-depletion was detected in Dp44mT-treated animals, probably because of the low doses of Dp44mT required to induce anti-tumor activity [23].
Previously, a number of studies were undertaken to characterize the mechanisms involved in the cytotoxicity of Dp44mT [21]. The electrochemistry of the iron complexes of the DpT analogues demonstrate facile interconversion between the iron(II) and iron(III) states, with the iron(III) complex retaining its oxidizing ability [21]. This was confirmed by the isolation of both the iron(II)- and iron(III)-DpT series complexes, indicating the stable nature of both the ferric and ferrous states [21]. The iron-Dp44mT redox potentials were found to lie within the range accessible to both cellular oxidants and reductants (E0 + 166 mV versus the normal hydrogen electrode) facilitating ROS generation under physiological conditions [21]. Also, the iron-complex of Dp44mT was shown to oxidize ascorbate and hydroxylate benzoate, again indicating the redox activity of the compound [21], [24], [25], [26]. Furthermore, some of the DpT ligands were found to induce iron-dependent OH-mediated strand-breaks in plasmid DNA [21].
Despite this previous work published in a number of studies [21], [24], [25], [26], the ability of the Dp44mT-iron complex to redox cycle and to produce OH has recently been questioned [27]. In fact, the Dp44mT-iron complex was shown by Hasinoff et al., using EPR spectroscopy not to induce OH formation [27]. Here, we put this claim to the test and demonstrate the opposite by using the same technique under very similar conditions [27]. We show that the Dp44mT-iron complexes are indeed redox-active and induce OH generation. In these studies, two different reaction conditions were used: (1) an iron(II)/H2O2 reaction system and (2) a reducing iron(III)/ascorbate reaction system in several different buffers, including Tris, phosphate or Hepes (10–20 mM, pH 7.4).
Section snippets
Spin-trap studies using DMPO
Many iron(III)-complexes, including those of EDTA, can be reductively activated to form the iron(II)-complex [28], which can produce OH or a high-valent iron-oxo species with OH-like reactivity, in a H2O2-dependent reaction [29]. The formation of OH can be demonstrated by the detection of methyl radical adducts, in EPR spin-trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with DMSO as a secondary spin-trap, rather than by formation of DMPO-OH, as this latter adduct can be
EPR: H2O2/Iron(II)–Dp44mT system
Initial investigations using EPR implemented the H2O2/iron(II)–Dp44mT system (Fig. 2). No signals were observed in solutions containing DMPO alone (Fig. 2A), or in Dp44mT/H2O2 samples lacking iron(II) (Fig. 2B). In contrast, H2O2/iron(II) with or without Dp44mT in the presence of DMPO led to the detection of OH, as evidenced by triplet of doublet signals, consistent with the formation of DMSO-derived, methyl radical adducts with DMPO (DMPO-CH3; aN 1.62 mT, aH 2.31 mT; Fig. 2C–F, H). The DMPO-CH3
Conclusions
Collectively, the current EPR study agrees with our previous investigations [21], [24], [25], [26], indicating that the Dp44mT-iron complex is redox-active and able to generate cytotoxic OH that can initiate oxidative damage to crucial biomolecules.
Acknowledgements
D.R.R. thanks the National Health and Medical Research Council of Australia (NHMRC) for Project Grant support and a Senior Principal Research Fellowship and the Prostate Cancer Foundation Australia for Concept Grant and Equipment Grant Support. D.R.R. D.B.L and P.J.J. appreciate grant support from the Australian Rotary Health Research Foundation. D.B.L thanks the Cancer Institute NSW for Fellowship support. C.L.H. thanks the NHMRC for Project Grant support and the National Heart Foundation of
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