Joan L. Buss

The Role of Iron Chelation in Cancer Therapy

This review focuses on advances and strategies in the use of iron chelators as anti-tumor therapies. Although the development of iron chelators for human disease has focused primarily on their use in the treatment of secondary iron overload, chelators may also be useful anti-tumor agents. They can deplete iron or cause oxidative stress in the tumor due to redox perturbations in its environment. Iron chelators have been tested for their anti-tumor activity in cell culture experiments, animal models and human clinical trials. Largely for pragmatic reasons, clinical studies of the anti-tumor activity of iron chelators have generally focused on desferrioxamine (DFO), a drug approved for the treatment of iron overload. These studies have shown that DFO can retard tumor growth in many different experimental contexts. However, the activity of DFO is modest, and advances in the use of chelators as anti-cancer agents will require the development of new chelators based on new paradigms. Examples of iron chelators that have shown promising anti-tumor activity (in various stages of development) include heterocyclic carboxaldehyde thiosemicarbazones, analogs of pyridoxal isonicotinoyl hydrazone, tachpyridine, O-trensox, desferrithiocin, and other natural and synthetic chelators. Apart from their use as single agents, chelators may also synergize with other anti-cancer therapies. The development of chelators as anticancer agents is largely an unexplored field, but one with extraordinary potential to impact human cancer.

Lipophilicity of analogs of pyridoxal isonicotinoyl hydrazone (PIH) determines the efflux of iron complexes and toxicity in K562 cells.

Iron overload secondary to beta-thalassemia and other iron-loading anemias is the most serious obstacle to be overcome in the treatment of these diseases, since there is no physiological mechanism for excretion of the excess iron acquired by chronic blood transfusion. Due to the inconvenience and cost of the current iron chelation therapy, the search for an orally available iron chelator is ongoing. Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective at mobilizing iron in vivo and in vitro at doses that are not toxic. PIH analogs were approximately equally effective at binding 59Fe within K562 cells; their efficacy depended upon the kinetics of release of the iron-chelator complex from the cell, which was correlated inversely with the lipophilicity of the chelators. Addition of BSA, which has a well-characterized affinity for lipophilic species, to the extracellular medium enhanced iron-chelator efflux, such that all analogs caused 59Fe release from the cells as quickly as it was chelated; this suggests that BSA acts as an extracellular sink for the iron-chelator complexes, many of which are highly lipophilic. The toxicity of the free chelators varied over two orders of magnitude, and was correlated with the amount of intracellular 59Fe-chelator complexes, implicating the complexes in the mechanism of toxicity of the chelators. Understanding the structural features that determine the efficacy and toxicity of iron chelators in biological systems is of value in the selection of PIH analogs for in vivo examination.

Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTA(CMV) (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTA(CMV) transgenes, FerH expression was increased 6.0+/-1.1-fold (mean+/-S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2(S)-S2(LAP) (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2+/-4.8-fold (mean+/-S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3+/-0.9-fold (mean+/-S.D.), concurrent with a 4.5+/-2.1-fold (mean+/-S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance.

The prolyl 4-hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate generates effective iron deficiency in cultured cells.

Ethyl‐3,4‐dihydroxybenzoate (EDHB) is commonly utilized as a substrate analog and competitive inhibitor of prolyl 4‐hydroxylases. These iron‐dependent enzymes have received a lot of attention for their involvement in crucial biochemical pathways such as collagen maturation and oxygen sensing. Since EDHB is also capable of chelating the enzyme‐bound iron, we study here its function as a chelator. We show that the affinity of EDHB for ferric iron is significantly lower than that of desferrioxamine. Nevertheless, EDHB is sufficient to promote effective iron deficiency in cells, reflected in the activation of the iron‐responsive element/iron regulatory protein regulatory network. Thus, treatment of B6 fibroblasts with EDHB results in slow activation of iron regulatory protein 1 accompanied by an increase in transferrin receptor levels and reduction of the ferritin pool.

Pyridoxal isonicotinoyl hydrazone analogs induce apoptosis in hematopoietic cells due to their iron-chelating properties

Analogs of pyridoxal isonicotinoyl hydrazone (PIH) are of interest as iron chelators for the treatment of secondary iron overload and cancer. PIH, salicylaldehyde isonicotinoyl hydrazone (SIH), and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (NIH), the toxicity of which vary over two orders of magnitude, were selected for a study of their mechanisms of toxicity. PIH analogs and their iron complexes caused concentration- and time-dependent apoptosis in Jurkat T lymphocytes and K562 cells. Bcl-2 overexpression was partially anti-apoptotic, suggesting mitochondrial mediation of apoptosis. Since the pan-caspase inhibitor zVAD-fmk did not reduce lysosomal and mitochondrial destabilization, these events occur upstream of caspase activation. In contrast, phosphatidylserine externalization and the development of apoptotic morphology were inhibited significantly, indicating the role of caspases in mediating these later events. Since overexpression of CrmA had no effect on apoptosis, caspase-8 is not likely involved. Fe(3+) complexes of SIH and NIH, which accumulated in 59Fe-labeled mouse reticulocytes during incubation with the chelators, also caused apoptosis. BSA, which promotes release of the complexes from cells, reduced the toxicity of SIH and NIH, suggesting that the induction of apoptosis by PIH analogs involves toxic effects mediated by their Fe(3+) complexes. Moreover, analogs of these agents lacking the iron-chelating moiety were non-toxic.

Mobilization of intracellular iron by analogs of pyridoxal isonicotinoyl hydrazone (PIH) is determined by the membrane permeability of the iron-chelator complexes

In the ongoing search for an effective, orally active iron-chelator, the capacity of a series of halogenated analogs of pyridoxal isonicotinoyl hydrazone (PIH) to bind intracellular 59Fe and cause its release from cells was investigated. Reticulocytes labeled with 59Fe(2)-transferrin in which heme synthesis was inhibited by succinylacetone were used as a model of 59Fe mobilization. The kinetics of iron binding were similar for all the chelators tested (half-time of approximately 1 hr), and all bound more than twice as much 59Fe as PIH. The rate of release of the 59Fe-chelator complexes from cells depended upon the structure of the chelators. Ortho-substituted analogs were more effective at mobilizing cellular iron than meta and para isomers, due to a more efficient release of the iron complexes from the cell. The iron-chelator complexes which were released slowly from cells had a high affinity for erythrocyte ghost membranes, indicating the role of membrane permeability in the release mechanism of the complexes. The addition of BSA to the extracellular medium increased the extent of iron release by lipophilic analogs in a concentration-dependent manner, presumably by acting as a sink for the lipophilic complexes. The affinity of BSA for the chelators and their Fe(3+) complexes, determined spectrophotometrically, demonstrated that all chelators and their iron complexes bound BSA with dissociation constants ranging from 7,000 to >500,000 M(-1). Understanding the importance of the rate of release of the iron-chelator complex will direct the search for iron-chelators with improved efficacy.

Alternative treatment paradigm for thalassemia using iron chelators.

OBJECTIVE beta-thalassemia major, or Cooleys anemia, is a red blood cell disorder requiring lifelong blood transfusions for survival. Erythrocytes accumulate toxic iron at their membranes, triggering an oxidative cascade that leads to their premature destruction in high numbers. We hypothesized that removing this proximate iron compartment as a primary treatment, using standard and alternative orally active iron chelators, could prevent hastened red cell removal and, clinically, perhaps alleviate the need for transfusion. MATERIALS AND METHODS Iron chelators of the pyridoxal isonicotinoyl hydrazone family (pyridoxal isonicotinoyl hydrazone and its analog pyridoxal ortho-chlorobenzoyl hydrazone) were evaluated in addition to the present mainstay, desferrioxamine and deferiprone, in vitro and in vivo. RESULTS Treatment of human beta-thalassemic erythrocytes with chelators resulted in significant depletion of membrane-associated iron and reduction of oxidative stress, as evaluated by methemoglobin levels. When administered to beta-thalassemic mice, iron chelators mobilized erythrocyte membrane iron, reduced cellular oxidation, and prolonged erythrocyte half-life. The treated thalassemic mice also showed improved hematological abnormalities. Remarkably, a beneficial effect as early as the erythroid precursor stage was manifested by normalized proportions of mature vs immature reticulocytes. All four compounds were also found to mitigate iron accumulation in target organs, a critical determinant for patient survival. In this respect, pyridoxal ortho-chlorobenzoyl hydrazone displayed higher activity relative to other chelators tested, further diminishing iron in liver and spleen by up to approximately fivefold and twofold, respectively. CONCLUSION Our study demonstrates the ability of iron chelators to improve several of the fundamental pathological disturbances of thalassemia, and reveals their potential for clinical use in diminishing requirement for transfusion when administered early in disease development.