Mechanisms underlying reductant-induced reactive oxygen species formation by anticancer copper(II) compounds

Abstract

Intracellular generation of reactive oxygen species (ROS) via thiol-mediated reduction of copper(II) to copper(I) has been assumed as the major mechanism underlying the anticancer activity of copper(II) complexes. The aim of this study was to compare the anticancer potential of copper(II) complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; currently in phase II clinical trials) and its terminally dimethylated derivative with that of 2-formylpyridine thiosemicarbazone and that of 2,2'-bipyridyl-6-carbothioamide. Experiments on generation of oxidative stress and the influence of biologically relevant reductants (glutathione, ascorbic acid) on the anticancer activity of the copper complexes revealed that reductant-dependent redox cycling occurred mainly outside the cells, leading to generation and dismutation of superoxide radicals resulting in cytotoxic amounts of H(2)O(2). However, without extracellular reductants only weak intracellular ROS generation was observed at IC(50) levels, suggesting that cellular thiols are not involved in copper-complex-induced oxidative stress. Taken together, thiol-induced intracellular ROS generation might contribute to the anticancer activity of copper thiosemicarbazone complexes but is not the determining factor.

Fig. 1

Overview of compounds investigated

Fig. 2

ORTEP plot of Cu-Triapine with thermal ellipsoids depicted at the 50% probability level (the H₃O⁺Cl⁻ fragment is omitted). Selected bond distances (Å) and angles (°): Cu–Cl, 2.2493(5); Cu–S, 2.281(3); Cu–N2, 1.937(9); Cu–N1, 2.031(8); S1–C7, 1.723(10); C7–N3, 1.311(13); N3–N2, 1.383(11); N2–C6, 1.317(13); N1–Cu–S, 164.5(2); N2–Cu–Cl, 176.9(3); N1–Cu–N2, 81.1(3); N2–Cu–S, 83.6(3)

Fig. 3

EPR spectrum of 1 mM Cu-Triapine in dimethyl sulfoxide (DMSO)/dimethyl formamide (DMF) (1:3 v/v) (black) and simulated spectrum (gray). Experimental conditions: X-band; temperature −196 °C, microwave frequency 9.4 GHz; microwave power 5 mW; modulation amplitude 2 G

Fig. 4

Cell death induction. The proportion of apoptotic cells (early and late apoptosis) was determined by Hoechst 33258/propidium iodide staining [44] after 24 h treatment with the indicated concentrations of the copper complexes tested. More than 500 cells from at least two samples for each concentration were analyzed and the percentages of early and late apoptotic cells at the indicated concentrations were determined by counting. One representative experiment of two giving comparable results is shown

Fig. 5

Intracellular reactive oxygen species (ROS) generation. Intracellular production of ROS in HL60 cells by the indicated concentrations of the copper complexes was determined after 30 min incubation using the ROS indicator 2′,7′-dichlorofluorescein diacetate (DCF-DA). Fluorescence was measured by flow cytometry. One representative experiment of three giving comparable results is shown. Significant differences were calculated by Student’s t test: single asterisk p < 0.05, two asterisks p < 0.01

Fig. 6

Impact of reductants on the anticancer activity of the copper complexes. To evaluate the effects of reductants, the glutathione (GSH) precursor N-acetylcysteine (NAC) and the antioxidant ascorbic acid (AA) were used. Briefly, after 30 min preincubation with NAC (1 and 2 mM) or AA (25 and 50 μM), SW480 cells were treated for 72 h with the indicated concentrations of the copper complexes. Viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The values given are the mean ± the standard deviation of three determinations from three experiments

Fig. 7

Reductant-induced ROS generation by the copper complexes. The influence of pretreatment with 2 mM NAC or 50 μM AA on the intracellular ROS levels in HL60 cells after incubation with the copper complexes (25 μM) was determined using the ROS indicator DCF-DA. Fluorescence was measured by flow cytometry. One representative experiment of three giving comparable results is shown. Significant differences were calculated by Student’s t test: two asterisks p < 0.01

Fig. 8

Impact of enhanced intracellular GSH levels on the anticancer activity of the copper complexes. a To evaluate the impact of elevated intracellular GSH levels on the copper complexes tested, SW480 cells were incubated with the GSH precursor NAC. After 30 min pretreatment, NAC-containing medium was replaced by fresh culture medium. Then the copper complexes were added at the indicated concentrations. After 72 h incubation, viability was determined using MTT assay. The values given are the mean ± the standard deviation of three determinations from three experiments. b Left DCF-DA-loaded HL60 cells were incubated for 15 min with 2 mM NAC, then the test compounds (50 μM) were added in the presence of NAC. After 30 min incubation, fluorescence was measured by flow cytometry. Right after preincubation, the NAC-containing buffer was replaced by fresh Hanks balanced salt solution and the test compounds (50 μM) were added. After 30 min incubation, fluorescence was measured by flow cytometry. One representative experiment of three giving comparable results is shown

Fig. 9

NAC- and AA-induced H₂O₂ production by the copper complexes. The dependence of the level of copper-complex-generated H₂O₂ on NAC (2 mM) and AA (50 μM) was determined using the xylenol orange-based PerOXOquant assay. The copper complexes were used at concentrations of 50 μM. The values given are the mean ± the standard deviation of three determinations

Fig. 10

Effect of extracellular superoxide dismutase (SOD) and catalase (CAT) on thiol-induced ROS generation. a Influence of SOD and CAT cotreatment (100 U/mL) on the NAC-induced (2 mM) ROS formation by Cu-Triapine (25 μM) in HL60 cells was determined using DCF-DA. Fluorescence was measured by flow cytometry. One representative experiment of three giving comparable results is shown. Significant differences were calculated by Student’s t test: two asterisks p < 0.01, three asterisks p < 0.001. b Left impact of CAT cotreatment (100 U/mL) on the anticancer activity of Cu-Triapine in the presence and absence of NAC. Right protective effects of CAT against H₂O₂-induced cell death. Cell viability was determined by MTT assay after 72 h drug treatment

Fig. 11

O₂^·− generation ability of the copper complexes in the presence of NAC and AA. The dependence of the level of copper-complex-generated O₂^·− on NAC (2 mM) and AA (50 μM) was determined by measuring the reduction of nitroblue tetrazolium spectrophotometrically. The copper complexes were used at concentrations of 25 μM. The values given are the mean ± the standard deviation of three determinations. Significant differences were calculated by Student’s t test: single asterisk p < 0.05, two asterisks p < 0.01

Fig. 12

Proposed extracellular redox reactions underlying the Cu-TSC-induced ROS generation