Abstract
Thiosemicarbazones such as Triapine (Tp) and Dp44mT are tridentate iron (Fe) chelators that have well-documented antineoplastic activity. Although Fe-thiosemicarbazones can undergo redox cycling to generate reactive species that may have important roles in their cytotoxicity, there is only limited insight into specific cellular agents that can rapidly reduce Fe(III)-thiosemicarbazones and thereby promote their redox activity. Here we report that thioredoxin reductase-1 (TrxR1) and glutathione reductase (GR) have this activity and that there is considerable specificity to the interactions between specific redox centers in these enzymes and various Fe(III) complexes. Site-directed variants of TrxR1 demonstrate that the selenocysteine (Sec) of the enzyme is not required, whereas the C59 residue and the flavin have important roles. Although TrxR1 and GR have analogous C59/flavin motifs, TrxR is considerably faster than GR. For both enzymes, Fe(III)(Tp)2 is reduced faster than Fe(III)(Dp44mT)2. This reduction promotes redox cycling and the generation of hydroxyl radical (HO) in a peroxide-dependent manner, even with low-micromolar levels of Fe(Tp)2. TrxR also reduces Fe(III)-bleomycin and this activity is Sec-dependent. TrxR cannot reduce Fe(III)-EDTA at significant rates. Our findings are the first to demonstrate pro-oxidant reductive activation of Fe(III)-based antitumor thiosemicarbazones by interactions with specific enzyme species. The marked elevation of TrxR1 in many tumors could contribute to the selective tumor toxicity of these drugs by enhancing the redox activation of Fe(III)-thiosemicarbazones and the generation of reactive oxygen species such as HO.