Figure 4

TPEN-Cu complex is redox active. A) Reduction of [TPEN-Fe ^(III)] and [TPEN-Cu ^(II)] by ascorbic acid. Reactions were carried out at 25°C in 0.1 M phosphate buffer (pH 7.4). TPEN, ferric nitrate, copper sulfate and ascorbic acid were used at concentrations of 0.2, 0.1, 0.1 and 5 mM, respectively. Complexes of TPEN with Cu ^(II) and Fe ^(III) were prepared via incubation of the ligand (0.3 mM) with the corresponding metal ion (0.03 mM) for 10 min in the reaction buffer. A) Changes in the absorbance of solutions of [TPEN-Fe^(III)] in the absence (open circles) and the presence (closed circles) of ascorbic acid. B) Electronic spectrum of TPEN (1) and [TPEN-Cu^(II)] in the absence (2) and the presence (3 – 5) of ascorbic acid. Consecutive spectra were recorded with time interval of 5 min. C-D) Fenton-like reactions of [TPEN-Cu^(II)] and [TPEN-Fe^(III)]. C) [TPEN-Cu^(II)], ascorbic acid, DMSO, H₂O₂ and PBN. D) [TPEN-Fe^(III)], ascorbic acid, DMSO, H₂O₂ and PBN. Reactions were carried out 25°C in 0.1 M phosphate buffer (pH 7.4). Ascorbic acid, DMSO, H₂O₂ and PBN were used at concentrations of 1 mM, 300 mM and 2 mM, respectively. Consecutive EPR spectra were recorded with time interval of 5 min. E) Concentrations of copper, zinc and iron in HCT116 and NCM460 (mean ± SD, n = 3). F) Percent copper chelated by TPEN in HCT116, Lovo and SW480 cells. Cells were treated with 5 μM TPEN for 24 h, and prepared cell lysates were filtered through 10 Kd cut off filtered to remove TPEN and TPEN-copper complexes. Copper was measured by atomic absorption in both the filtrate (TPEN Bound) and the lysates (Protein Bound) and percentages calculated accordingly (mean ± SD, n = 3).