Disulfide-masked iron prochelators: Effects on cell death, proliferation, and hemoglobin production

Abstract

The iron metabolism of malignant cells, which is altered to ensure higher acquisition and utilization, motivates the investigation of iron chelation strategies in cancer treatment. In a prochelation approach aimed at increasing intracellular specificity, disulfide reduction/activation switches are incorporated on iron-binding scaffolds resulting in intracellularly activated scavengers. Herein, this strategy is applied to several tridentate donor sets including thiosemicarbazones, aroylhydrazones and semicarbazones. The novel prochelator systems are antiproliferative in breast adenocarcinoma cell lines (MCF-7 and metastatic MDA-MB-231) and do not result in the intracellular generation of oxidative stress. Consistent with iron deprivation, the tested prochelators lead to cell-cycle arrest at the G₁/S interface and induction of apoptosis. Notably, although hemoglobin-synthesizing blood cells have the highest iron need in the human body, no significant impact on hemoglobin production was observed in the MEL (murine erythroleukemia) model of differentiating erythroid cells. This study provides new information on the intracellular effects of disulfide-based prochelators and indicates aroylhydrazone (AH1-S)₂ as a promising prototype of a new class of antiproliferative prochelator systems.

Keywords: Anticancer; Cell-cycle arrest; Hemoglobin production; Iron; Prochelator.

Fig. 1

Examples of iron-binding units (in red) in chelators employed for biomedical applications.

Fig. 2

Disulfide-based prochelators and thioether control compound employed in this study.

Fig. 3

Assessment of intracellular generation of ROS upon incubation with selected prochelators. MDA-MB-231 cells were treated with the indicated compounds (50 μM, 2 h), washed, treated with DCFH₂-DA (30 μM, 30 min) in PBS, and then analyzed by flow cytometry. Hydrogen peroxide is used as a positive control and SIH as a negative control. Values are presented as mean ± SDM (n=3), ** p <0.01.

Fig. 4

Effect of prochelators on cell cycle in MDA-MB-231 cells. Cells were treated with the compounds (10 or 50 μM, 12 h), harvested, fixed, pelleted and then treated with RNAse and propidium iodide (0.5 mg/mL and 40 μg/mL, respectively, 30 min) prior to analysis by flow cytometry. Values are presented as mean ± SDM (n=3), * p < 0.05 and ** p < 0.01.

Fig. 5

Investigation of cell death in the presence of disulfide-based prochelators. Jurkat cells were incubated with the tested compounds (20 μM, 48 h) or vehicle only (DMSO, untreated control). Following treatment with FTIC-AnnV and propidium iodide (PI), the cells were analyzed by flow cytometry. Values are presented as mean ± SDM (n=3), * p <0.05 and ** p <0.01.

Fig. 6

Effect of iron prochelators on intracellular haemoglobin (Hb) concentrations in the MEL model of erythropoiesis. Hb content was assessed after treatment with prochelators alone (top) or along with succinylacetone (SA, 1 mM). Values (mean ± SDM, n=3) are normalized to the untreated control (0 μM), * p <0.05 and ** p <0.01.

Scheme 1

Reduction/activation of a disulfide switch in thiosemicarbazone prochelator (TC1-S)₂