Iron metabolism, chelation and disease

Organisms depend on iron to survive. This fact is underscored by the critical requirement for iron during DNA synthesis and as a cofactor in proteins involved in respiration and oxygen transport. However, when present in excess of cellular requirements, iron can be toxic, due to its ability to generate reactive oxygen species and induce oxidative stress. The physiological significance of iron renders it a target for the development of iron chelators as therapeutic agents and highlights the potential problems that can occur when iron regulatory pathways are disturbed in disease. The rapid rate of neoplastic cell replication and the involvement of iron in cell cycle progression and DNA synthesis, highlight the potential for using iron chelators for cancer treatment. Chapter 3 of this thesis demonstrates the broad-spectrum in vitro and in vivo anti-tumour activity of the novel iron chelator, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) (Whitnall et.al., Proc Natl Acad Sci USA 2006; 103:14901-6). In vitro results illustrate the potency of Dp44mT over the clinically used chemotherapeutic agent, doxorubicin, and the ability of Dp44mT to overcome multi­drug resistance. The unique ability of Dp44mT to up-regulate the tumour growth and metastasis suppressor, Ndrgl in in vivo experiments, may account for this ligands selective anti-tumour activity (Whitnall et al., 2006). Collectively, these studies demonstrate that iron chelators such as Dp44mT, may be valuable anti-cancer compounds, particularly considering the emergence of multi-drug resistance in tumours. There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich 's ataxia (FA). The identification of potentially toxic mitochondrial iron deposits in FA suggests iron plays a role in its pathogenesis and merits the use of iron chelation therapy for the treatment of FA. Studies in Chapters 4 and 5 used the muscle creatine kinase (MCK) frataxin mutant mouse model that reproduces the classical traits associated with cardiomyopathy in FA, to study the molecular alterations which underlie the pathogenesis of this disease and assess the use of iron chelation therapy (Whitnall et.al., Proc Natl Acad Sci USA 2008; 105:9757-62). Studies specifically in Chapter 4 show that the increased mitochondrial iron in the myocardium of mutants was due to marked transferrin-iron uptake, which was the result of enhanced transferrin receptor 1 (TfRl) expression. 1n contrast to the mitochondrion, cytosolic ferritin expression and the proportion of cytosolic iron were decreased in mutant mice, indicating the cytosol was iron deficient. These studies demonstrate that loss of frataxin alters cardiac iron metabolism due to pronounced changes in iron trafficking away from the cytosol to the mitochondrion. Further work in Chapter 4 showed that the mitochondrial-permeable ligand, pyridoxal isonicotinoyl hydrazone, in combination with the hydrophilic chelator, desferrioxamine, prevented cardiac iron loading and limited cardiac hypertrophy in mutants, but did not lead to overt cardiac iron depletion or toxicity (Whitnall et al., 2008). However, iron chelation did not prevent decreased succinate dehydrogenase expression in the mutants nor loss of cardiac function, indicating that frataxin function must also be replaced in addition to removing the excess mitochondrial iron. In summary, for the first time, studies in this thesis demonstrate that frataxin deficiency markedly alters cellular iron trafficking and that iron chelation limits myocardial hypertrophy in the MCK mutant model of FA. To address the cytosolic iron deficiency in the cardiomyocytes of mutant mice, in Chapter 5, mice were fed a high iron diet aimed at reconstituting the iron deprived cytosolic compartment. From these studies, a significant decrease in cardiac hypertrophy was observed in high iron diet fed mice. Interestingly, while wild-type (WT) mice responded to the high iron diet by decreasing cardiac TfRl expression, no such compensation was observed in high-compared to normal-iron iron diet fed mutants. Similarly, activity of iron regulatory protein 2 (i.e., IRP2 RNA-binding activity) was not decreased in high iron diet fed mutants. These findings demonstrate the mutant heart does not respond to increased iron levels as does the WT animal. An intriguing and important outcome of dietary iron loading investigations, was the marked increase in iron concentration observed in the liver, spleen and kidney of mutant mice that were fed a normal iron diet. The MCK mutant mouse experiences deletion of frataxin in the heart only, and hence, the increase in iron levels observed in frataxin ­intact tissues such as the liver, indicated that the heart is able to influence systemic iron metabolism. Supporting this, changes were observed in iron-metabolism proteins such as hemojuvelin and TfRl not only in the heart, but in the liver. Collectively, these results indicate that frataxin knockout in the heart and the alterations in iron metabolism which lead to cytosolic iron deficiency in the heart, activate a systemic signalling mechanism, most likely to communicate its need for iron within the cytosolic compartment. In the final section of Chapter 5, transmission electron microscopy and magnetic susceptibility measurements were used to assess the molecular composition of accumulated iron in the MCK mutants. These studies showed that the iron accumulating in the mutant heart is not present within ferritin, but in well crystallised anti­ferromagnetic mineral aggregates. In conclusion, the investigations described within this thesis demonstrate the potential for iron chelators to be used for the treatment of cancer and FA. Moreover, they also begin to elucidate the marked alterations in the pathways of iron metabolism that occur on both a cellular and systemic level in FA. ln terms of contributing to our understanding of basic physiological iron homeostasis, they also identify that cardiac iron status is able to markedly influence systemic iron metabolism