Methyl and ethyl ketone analogs of salicylaldehyde isonicotinoyl hydrazone: Novel iron chelators with selective antiproliferative action

https://doi.org/10.1016/j.cbi.2012.03.010Get rights and content

Abstract

Salicylaldehyde isonicotinoyl hydrazone (SIH) is a lipophilic, orally-active tridentate iron chelator providing both effective protection against various types of oxidative stress-induced cellular injury and anticancer action. However, the major limitation of SIH is represented by its labile hydrazone bond that makes it prone to plasma hydrolysis. Recently, nine new SIH analogues derived from aromatic ketones with improved hydrolytic stability were developed. Here we analyzed their antiproliferative potential in MCF-7 breast adenocarcinoma and HL-60 promyelocytic leukemia cell lines. Seven of the tested substances showed greater selectivity than the parent agent SIH towards the latter cancer cell lines compared to non-cancerous H9c2 cardiomyoblast-derived cells. The tested chelators induced a dose-dependent dissipation of the inner mitochondrial membrane potential, an induction of apoptosis as evidenced by Annexin V positivity or significant increases of activities of caspases 3, 7, 8 and 9 and cell cycle arrest. With the exception of nitro group-bearing NHAPI, the studies of iron complexes of the chelators confirmed the crucial role of iron in the mechanism of their antiproliferative action. Finally, all the assayed chelators inhibited the oxidation of ascorbate by iron ions indicating lack of redox activity of the chelator–iron complexes. In conclusion, this study identified several important design criteria for improvement of the antiproliferative selectivity of the aroylhydrazone iron chelators. Several of the novel compounds – in particular the ethylketone-derived HPPI, NHAPI and acetyl-substituted A2,4DHAPI – merit deeper investigation as promising potent and selective anticancer agents.

Highlights

► New analogs of aroylhydrazone iron chelator SIH examined for antiproliferative action. ► Higher toxicity observed towards the cancer cell lines compared to non-malignant cells. ► Dissipation of mitochondrial membrane potential, apoptosis, cell cycle arrest. ► Ethylketone-derived HPPI and nitro group-bearing NHAPI possessed the best properties.

Introduction

Iron (Fe) is an essential micronutrient for cellular proliferation. As part of the Fe-containing proteins it catalyses many biochemical reactions of vital importance for energy metabolism, cellular respiration or DNA synthesis [1], [2], [3]. Because of their uncontrolled proliferation, cancer cells have higher Fe requirement. Various studies demonstrated significantly increased Fe levels in malignant tumors compared to healthy tissue [4], [5]. In breast cancer, higher levels of Fe were associated with increased aggressiveness which may be related to enhanced expression of transferrin receptors (TfR) on the cell surface [6]. Indeed, estrogen stimulation of estrogen receptor-positive breast cancer cells results in higher transcription of TfR genes [7], [8]. In addition, it has been recently demonstrated that the excretion of Fe from breast cancer cells is decreased due to the reduction of excreting protein ferroportin [9]. Similarly, studies on acute lymphoblastic [10] or chronic granulocytic leukemia [11] noticed an increased level of serum Fe, which was significantly decreased during a remission of the disease. More recent evidence suggests that Fe chelators have considerable potential to inhibit the proliferation of leukemic cells [12], [13], [14] and also to induce their differentiation [15].

Salicylaldehyde isonicotinoyl hydrazone (SIH) is a tridentate aroylhydrazone chelator forming selectively 2:1 complexes with both Fe3+ and Fe2+ ions [16], [17]. Due to its small molecule and sufficient lipophilicity, SIH can be administered orally. Compared with other aroylhydrazones such as pyridoxal isonicotinoyl hydrazone (PIH) or 1-naphthaldehyde isonicotinoyl hydrazone (NIH), SIH has the most favorable lipophilic–hydrophilic properties for passing through membranes and that is why SIH is highly effective in transporting Fe ions away from cells [18]. Reported biological properties of SIH include the protection of guinea-pig or neonatal rat isolated cardiomyocytes, rat cardiomyoblast cell line H9c2 and retinal cell line ARPE-19 against the H2O2-induced cell injury [19], [20], [21], [22]. The good effectiveness of SIH was also noted in protection of H9c2 cardiomyoblasts against oxidative injury induced by tert-butyl hydroperoxide and catecholamines and/or their reactive intermediates [23], [24]. In addition, it has been recently shown as an effective radioprotective agent [25] and possessed protective potential at a mouse model of amyotrophic lateral sclerosis [26]. The treatment with Fe–SIH complexes has reduced the expression of viral proteins and RNA of hepatitis C virus [27]. High potential of this compound to protect against the cardiotoxicity of anthracycline daunorubicin is another positive effect of SIH. This was demonstrated both in vitro with isolated neonatal rat cardiomyocytes [28] and in vivo using chronic heart failure model in rabbits [29]. Interestingly, the in vitro study revealed that Fe chelation with SIH differently modulated the anthracycline toxicity to cardiac and cancer cells. At concentrations when SIH significantly reduced the daunorubicin toxicity to rat cardiomyocytes, it augmented the antiproliferative action of daunorubicin in HL-60 acute promyelocytic leukemia cell line [28]. Finally, low in vivo toxicity and good tolerability of SIH has been demonstrated following its 10-week repeated administration to rabbits [30].

Despite these promising pharmacodynamic properties, the pilot pharmacokinetic study of SIH has revealed rather short biological half-life of this substance following its single intravenous administration to rabbits, apparently due to its labile hydrazone bond which makes it prone to hydrolysis [31], [32], [33]. This property is apparently inherent for all aroylhydrazones derived from aromatic aldehydes [32]. Recently, new SIH analogs derived from aromatic ketones significantly more resistant against the hydrazone bond hydrolysis were designed and synthesized (Fig. 1) [34].

The aim of this study was to examine the toxic effects of these agents against proliferating cancer cells and compare them with SIH and a well-known siderophore deferoxamine (DFO) as reference agents. MCF-7 human breast adenocarcinoma and HL-60 human promyelocytic leukemia cells were used in this study as they represent in vitro models of both solid tumors and hematological malignancies. Selectivity of their toxicity against cancer cells was assessed by comparison with their toxicities towards non-cancerous H9c2 cardiomyoblasts. Following the initial screening, SIH and several promising new chelators with better selectivity towards cancer cells were further examined for their effects on cell cycle, mitochondrial destabilization and induction of apoptosis. More general aim of this work was to identify the design criteria for future improvement of antiproliferative potency of aroylhydrazone iron chelators.

Section snippets

Chemicals

Salicylaldehyde isonicotinoyl hydrazine (SIH) was synthesized as described previously [35]. (E)-N′-[1-(2-hydroxyphenyl)ethyliden] isonicotinoylhydrazide (HAPI), (E)-N′-[1-(2-hydroxyphenyl)propyliden] isonicotinoylhydrazide (HPPI), (E)-N′-[1-(2,4-dihydroxyphenyl)ethyliden] isonicotinoylhydrazide (2,4DHAPI), (E)-N′-[1-(2,6-dihydroxyphenyl)ethyliden] isonicotinoylhydrazide (2,6DHAPI), (E)-N′-[1-(2-hydroxy-4-methoxyphenyl)ethyliden] isonicotinoylhydrazide (MHAPI), (E)-N

Antiproliferative effects of Fe chelators

To assess the antiproliferative/cytotoxic effects of the studied chelators, cells were incubated with different concentrations of the aroylhydrazones for 72 h. MCF-7 human adenocarcinoma cell line was used as a model of solid tumors, while HL-60 human promyelocytic leukemia cell line served as a model of hematological malignity. The toxic effects of the chelators were also examined on the non-cancerous H9c2 cardiomyoblast cell line in order to assess degree of selectivity of their toxic actions

Discussion

Iron chelators have been successfully used in clinical practice for management of diseases associated with chronic body Fe overload, such as β-thalassemia major [40], [41]. In these diseases they promote Fe excretion and act as protectors against oxidative injury caused by free redox-active Fe – the key catalyst in the Fenton chemistry [42]. However, in the last decades, a new possible use of Fe chelators in medicine emerged as they have shown great potential in anticancer treatment [40], [41].

Conflict of interest statement

The authors declare no competing financial interests.

Acknowledgement

This study was supported by the Charles University (Projects GAUK 299511, SVV 265001, 265004, UNCE 33/2012).

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