Identification of the di-pyridyl ketone isonicotinoyl hydrazone (PKIH) analogues as potent iron chelators and anti-tumour agents

Abstract

(1) In an attempt to develop chelators as potent anti-tumour agents, we synthesized two series of novel ligands based on the very active 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) group. Since lipophilicity and membrane permeability play a critical role in Fe chelation efficacy, the aldehyde moiety of the PCIH series, namely 2-pyridylcarboxaldehyde, was replaced with the more lipophilic 2-quinolinecarboxaldehyde or di-2-pyridylketone moieties. These compounds were then systematically condensed with the same group of acid hydrazides to yield ligands based on 2-quinolinecarboxaldehyde isonicotinoyl hydrazone (QCIH) and di-2-pyridylketone isonicotinoyl hydrazone (PKIH). To examine chelator efficacy, we assessed their effects on proliferation, Fe uptake, Fe efflux, the expression of cell cycle control molecules, iron-regulatory protein-RNA-binding activity, and (3)H-thymidine, (3)H-uridine and (3)H-leucine incorporation. (2) Despite the high lipophilicity of the QCIH ligands and the fact that they have the same Fe-binding site as the PCIH series, surprisingly none of these compounds were effective. In contrast, the PKIH analogues showed marked anti-proliferative activity and Fe chelation efficacy. Indeed, the ability of these ligands to inhibit proliferation and DNA synthesis was similar or exceeded that found for the highly cytotoxic chelator, 311. In contrast to the PCIH and QCIH analogues, most of the PKIH group markedly increased the mRNA levels of molecules vital for cell cycle arrest. (3) In conclusion, our studies identify structural features useful in the design of chelators with high anti-proliferative activity. We have identified a novel class of ligands that are potent Fe chelators and inhibitors of DNA synthesis, and which deserve further investigation.

Figure 1

Schematic illustration showing the structure of the cytotoxic Fe chelator, 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), compared to the three groups of aroylhydrazone chelators assessed in this study: 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), di-2-pyridylketone isonicotinoyl hydrazone (PKIH), and 2-quinolinecarboxaldehyde isonicotinoyl hydrazone (QCIH).

Figure 2

Schematic illustration showing the three groups of aroylhydrazone chelators, the substituents (R1), and the nomenclature used to identify each chelator.

Figure 3

The effect of the chelators on ⁵⁹Fe mobilization from prelabelled SK-N-MC neuroepithelioma cells. (A) Comparison of the PKIH and PCIH ligands; (B) Comparison of the QCIH and PCIH ligands. Cells were labelled with ⁵⁹Fe-transferrin (0.75 μM) for 3 h at 37°C, washed, and then reincubated for 3 h at 37°C in the presence of medium alone (control) or medium containing DFO (100 μM) or the other chelators (50 μM). Results are expressed as the mean±s.d. of three replicates in a typical experiment of two experiments performed.

Figure 4

The effect of the chelators on ⁵⁹Fe uptake from ⁵⁹Fe-transferrin (⁵⁹Fe-Tf) by SK-N-MC neuroepithelioma cells. (A) Comparison of the PKIH and PCIH ligands; (B) Comparison of the QCIH and PCIH ligands. The cells were incubated for 3 h at 37°C in media containing ⁵⁹Fe-Tf (0.75 μM) and either DFO (100 μM) or the other chelators (50 μM), washed, and then incubated with pronase (1 mg ml) for 30 min at 4°C. Results are expressed as the mean±s.d. of three replicates in a typical experiment of two experiments performed.

Figure 5

The effect of concentration of the PKIH analogues compared to the highly active PCTH chelator on: (A) cellular ⁵⁹Fe mobilization and (B) internalized ⁵⁹Fe uptake from ⁵⁹Fe-transferrin (⁵⁹Fe-Tf). (A) Cells were labelled with ⁵⁹Fe-Tf (0.75 μM) for 3 h at 37°C, washed, and then reincubated for 3 h at 37°C in the presence of medium alone (control) or medium containing the chelators (0.5–50 μM). (B) The cells were incubated for 3 h at 37°C in media containing ⁵⁹Fe-Tf (0.75 μM) alone (control) or with the chelators (0.5–50 μM), washed, and then incubated with pronase (1 mg ml) for 30 min at 4°C. Results are expressed as the mean±s.d. of three replicates in a typical experiment of two experiments performed.

Figure 6

The influence of one of the most effective PKIH analogues (PKIH) compared to desferrioxamine (DFO) on the proliferation of SK-N-MC neuroepithelioma cells (N) compared to MRC-5 fibroblasts (F). Cells were incubated in the presence and absence of the chelators (0–25 μM) for 72 h at 37°C. After this incubation period, cellular density was measured via the MTT assay (see Methods for details). Each data point represents the mean of three separate experiments with duplicate determinations in each experiment.

Figure 7

Many of the PKIH series of Fe chelators markedly increase WAF1 and GADD45 mRNA expression in SK-N-MC neuroepithelioma cells. The effect of the PKIH group of chelators are compared to DFO, 311 and some chelators of the PCIH series. (A) (i) Ethidium bromide staining of the agarose gel, (ii) GADD45, (iii) WAF1, and (iv) β-actin mRNA levels; (B) Densitometric analysis of the results in (A) normalized to the β-actin loading control. Total RNA was extracted from cells after a 20 h incubation at 37°C with medium alone (control) or medium containing DFO (100 μM) or the other chelators (25 μM). Northern blotting was performed using standard procedures (see Methods). The result illustrated is a typical experiment from three experiments performed.

Figure 8

All of the PKIH analogues increase iron-regulatory protein (IRP)-RNA-binding activity in SK-N-MC neuroepithelioma cells. (A) Active IRP-RNA-binding activity; (B) Densitometric analysis of the results in (A). The effect of the PKIH analogues were compared to several chelators that acted as internal controls (ie., DFO, 311, PCBBH, and PCAH) and also the Fe donor, ferric ammonium citrate (FAC). Cells were incubated with DFO (100 μM), FAC (100 μg ml) and the remaining chelators (25 μM) for 20 h at 37°C. The IRP-RNA-binding activity was then assessed by the gel-retardation assay using standard techniques (see Methods). The result illustrated is a typical experiment from three experiments performed.