Lipophilicity of analogs of pyridoxal isonicotinoyl hydrazone (PIH) determines the efflux of iron complexes and toxicity in K562 cells
Introduction
The importance of iron in biological systems is due mainly to its roles in oxygen distribution and electron transfer. Iron in excess of the capacity of the organism to use or store it is toxic, presumably via formation of reactive oxygen species, including the extremely reactive hydroxyl radical [1], which cause oxidative stress. To prevent uncontrolled redox reactions, and to conserve a poorly bioavailable metal, iron metabolism in mammals is tightly regulated, and involves efficient recycling [2]. Because there is no physiological mechanism of iron excretion, patients receiving chronic blood transfusions develop iron overload, the current treatment for which is desferrioxamine, a drug that must be administered via subcutaneous infusion [3] due to its very short plasma half-time [4].
An orally effective iron chelator is urgently needed as a convenient and inexpensive alternative to desferrioxamine therapy. The efficacy of PIH (Fig. 1) in iron mobilization has been characterized in vitro[5] and in vivo[6]. Screens of PIH analogs using -labeled cell culture models have identified several chelators in this series that are more active than PIH [7], [8], [9], including pyridoxal para-methoxybenzoyl hydrazone, pyridoxal meta-chlorobenzoyl hydrazone (m-ClPBH), and pyridoxal meta-fluorobenzoyl hydrazone (m-FPBH). These analogs also mobilized more in rats than PIH, whether administered intraperitoneally or orally [10]. A recent study has demonstrated that the property defining the capacity of PIH analogs to mobilize from reticulocytes is the rate of efflux of the iron–chelator complexes from the cells [11], presumably via passive diffusion. It is of interest whether these chelators mobilize similarly from other cell lines, which, as compared with reticulocytes, may be expected to have different intracellular pathways of iron trafficking.
Several studies have demonstrated the antiproliferative effects of some PIH analogs in cell culture models [8], [12], [13], [14], [15]. It has been demonstrated that mobilization and inhibition of DNA synthesis were not correlated [16], suggesting that iron depletion from cells, which is expected to limit the iron available to ribonucleotide reductase, is insufficient to account for the antiproliferative effects of these chelators. Hence, it is of value to examine other mechanisms by which the toxicity of PIH analogs may be mediated.
Section snippets
Synthesis of PIH analogs
Pyridoxal hydrochloride was purchased from Sigma. Salicylaldehyde and 2-hydroxy-1-naphthylaldehyde were purchased from Aldrich. Isonicotinic acid hydrazide and benzoyl hydrazide were purchased from Lancaster. The halogenated acid benzoyl hydrazides used for preparation of the hydrazones were purchased from Transworld Chemicals. PIH and its analogs were synthesized according to standard methods as previously described [17]. All other materials were of the highest quality available.
Preparation of chelator and Fe(chelator)2 solutions
Stock
59Fe mobilization from K562 cells
K562 cells were labeled with by incubation for 3 hr with 2–transferrin as described in Section 2. After this period of incubation, is distributed between ferritin, the major iron storage protein, and the poorly characterized LIP [15]. The steps involved in mobilization, defined in Fig. 2, were examined separately after the incubation of cells with the chelators by measuring the radioactivity in the incubation medium and in an ethanol-soluble cytoplasmic fraction, which
59Fe mobilization in K562 cells by chelators
PIH analogs were effective in mobilizing from K562 cells, many of which were more effective than PIH itself (Fig. 6). At the concentrations used in this study, all chelators bound with similar kinetics (Fig. 3, Fig. 4), but the kinetics of release from the cells varied markedly. The maximum amount of mobilized from K562 cells by 100 μM PIH analogs depended primarily upon the kinetics of release of –chelator complexes from the cells (Fig. 3, Fig. 4, Fig. 6), which is
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