Concentration of Fe(3+)-Triapine in BEAS-2B Cells

Abstract

An electron paramagnetic resonance (EPR) method was used to determine the concentration of the antitumor agent Triapine in BEAS-2B cells when Triapine was bound to iron (Fe). Knowledge of the concentration of Fe-Triapine in tumor cells may be useful to adjust the administration of the drug or to adjust iron uptake in tumor cells. An EPR spectrum is obtained for Fe(3+)-Triapine, Fe(3+)(Tp)₂⁺, in BEAS-2B cells after addition of Fe(3+)(Tp)₂⁺. Detection of the low spin signal for Fe(3+)(Tp)₂⁺ shows that the Fe(3+)(Tp)₂⁺ complex is intact in these cells. It is proposed that Triapine acquires iron from transferrin in cells including tumor cells. Here, it is shown that iron from purified Fe-transferrin is transferred to Triapine after the addition of ascorbate. To our knowledge, this is the first time that the EPR method has been used to determine the concentration of an iron antitumor agent in cells.

Keywords: BEAS-2B bronchial epithelial cells; EPR; Triapine; electron paramagnetic resonance.

Figure 1

Schematic of FeTp₂ designated Fe-Triapine. Reprinted with permission from Kowol, C.R.; Trondl, R.; Heffeter, P.; Arion, V.B.; Jukupec, M.A.; Roller, A., Galanski, M.; Berger, W.; Keppler, B.K. Impact of metal coordination on cytotoxicity of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine) and novel insights into terminal dimethylation, J. Med. Chem., 2009, 52, 5032–5043, doi: 10.1021/jm900528d). Copyright 2009 American Chemical Society.

Figure 2

The electron paramagnetic resonance (EPR) spectra of BEAS-2B cells (6 × 10⁷ cells/mL) treated with Fe(3+)(Tp)₂⁺ (33 µM concentration in cells). Insert: The EPR signal for Fe(3+)(Tp)₂⁺ at different concentrations. Spectrometer conditions, 5 G mod.; microwave freq., 9.633 GHz; 7 K; 25 scans; microwave power, 0.2 mW.

Figure 3

EPR spectrum for 80 µM FeTf with or without 30 µM Triapine (top spectrum) and FeTf plus Triapine plus ascorbate (bottom spectrum). The addition of ascorbic acid to FeTf in the presence of Triapine results in a decrease in the FeTf signal and the appearance of the FeT₂⁺ signal.

Figure 4

Reaction of Fe(2+)-Triapine, Fe(2+)(Tp)₂, with oxygen to generate superoxide (O₂^●−), H₂O₂, and hydroxyl radical. Bottom: Coupled reactions for thioredoxin reductase (TrxR), thioredoxin (Trx), thioredoxin-2 (Trx2), and perioxiredoxin-3 (Prx3). Note: red = reduced, ox = oxidized.