Competing pathways of iron chelation: Angiogenesis or anti‐tumor activity: Targeting different molecules to induce specific effects (2004)

In a recent letter published in the International Journal of Cancer, Linden and Wenger1 commented on the anti-proliferative and anti-angiogenic activity of ciclopirox (CPX) that was reported in a previous article.2 Linden and Wenger1 point out that CPX is an anti-fungal agent that binds iron (Fe) and inhibits tumor growth in vitro by this mechanism.3 Moreover, the Fe-depletion induced by this compound can also encourage angiogenesis by upregulating the vascular endothelial growth factor-1 (VEGF-1).1 This effect is mediated through the stabilization of hypoxia inducible factor-1α (HIF-1α) via inhibiting proline hydroxylation of this protein by an Fe-containing prolyl-4-hydroxylase.1 Indeed, Linden and Wenger1 cite work by Simonart et al.4 who implemented the clinically used Fe chelator, desferrioxamine (DFO), to treat immunodeficient mice implanted with the endothelial-derived tumor, Kaposi's sarcoma. In this latter investigation, DFO did not inhibit growth of the cancer and resulted in slightly larger tumors in vivo compared to animals treated with vehicle alone.4 Based on these studies, Linden and Wenger1 suggest that CPX may not be suited toward the treatment of cancer in vivo but could possibly be used to increase angiogenesis for wound healing. Although the point of Linden and Wenger1 is clearly of interest, we would like to suggest that the chelator-mediated response obtained, that is either angiogenesis or anti-proliferative activity, are dependent on the extent of Fe chelation and the intracellular pathways targeted by these ligands. Indeed, as discussed below, both of these competing pathways can be induced by Fe chelation and it may be possible to pharmacologically promote either pathway depending upon the design of the ligand. A critical consideration in terms of the balance between inducing angiogenesis or anti-proliferative activity is the efficacy of Fe chelation. For example, DFO is a relatively poor anti-proliferative agent due to its short plasma half-life and its inability to readily permeate cell membranes to bind intracellular Fe pools.5, 6 In fact, the cited study of Simonart et al.3 clearly demonstrated that DFO did not induce Fe-depletion, as reflected by the lack of alteration in serum ferritin levels, serum Fe levels, total Fe-binding capacity, transferrin-Fe saturation or the hematocrit. Hence, the lack of effect on tumor growth was not surprising, as the compound was ineffective at inducing Fe-depletion. Indeed, the concentration of DFO used in the Simonart et al.3 study may be insufficient to result in Fe deprivation and local toxicity. As a consequence of the poor anti-proliferative activity and Fe chelation efficacy of DFO, several laboratories have developed novel Fe chelators that have high membrane permeability and marked anti-proliferative activity compared to DFO.5, 6 For example, there are multiple Fe chelators that cause marked cell cycle arrest and apoptosis in vitro and in vivo, including desferrithiocin analogs, tachpyridine, O-trensox, pyridoxal isonicotinoyl hydrazone (PIH) analogs and Triapine®.5, 6 In fact, Triapine® is currently in clinical trials for the treatment of a variety of cancers.7, 8 Importantly, although the treatment of tumors with potent Fe chelators results in HIF-1α stabilization and VEGF-1 expression to possibly promote angiogenesis,1, 9 it must be pointed out that there are also counteracting pathways induced by Fe-depletion that inhibit growth. In fact, effective Fe chelation is well known to cause cell cycle arrest and apoptosis.5, 6, 9 For instance, without a constant supply of Fe, the conversion of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase is inhibited and DNA synthesis prevented.5, 6 In addition, recent studies have demonstrated that Fe chelators decrease the expression of molecules required for cell cycle progression such as the cyclins (e.g., cyclin A, B, D) and cdk2.10 To further enforce cell cycle arrest and programmed cell death, Fe chelators inhibit the translation of p21WAF1/CIP1.11 Concerning all Fe chelation studies, it is important to distinguish between the toxicity results obtained in vitro and in vivo. In vitro, there is a concentration window where Fe chelators show low toxicity against normal cells, but high cytotoxicity toward tumors. In contrast, the situation is more complex in vivo, and as a consequence, extrapolations from in vitro results can be misleading. This is especially relevant for chelation therapy using potent Fe chelators because the majority of toxicity studies have been carried out in vitro. It is important to note, however, that several investigations have demonstrated the effectiveness of Fe chelation in vivo using DFO and Triapine,5, 6, 7, 8 thus encouraging further studies. The multiple mechanisms by which potent Fe chelators cause cell cycle arrest and programmed cell death may overwhelm the angiogenic effects of VEGF-1 and account for the anti-tumor activity of chelators in vivo and in many clinical trials.5, 6 Furthermore, it is known that the upregulation of HIF-1α by potent Fe chelators diverges from the angiogenic pathway to induce apoptosis.9, 10 In fact, a study by Furukawa et al.12 using DFO showed HIF-1α-dependent upregulation of the PLAGL2 transcription factor that transactivates Nip3 to cause cell death.12, 13 This principle has been demonstrated recently using the potent mycobacterial Fe chelator, desferri-exochelin.14 Incubation with this chelator increased the expression of both VEGF-1 and Nip3 and prevented the effects of the former protein on endothelial cell proliferation.14 Furthermore, the ligand was also shown to cause tumor cell death via inducing apoptosis.15 It is also interesting to note that there are studies that suggest Fe is required for angiogenesis. Indeed, Carlevaro et al.16 demonstrated that the Fe-transport molecule, transferrin, is a major angiogenic stimulus released by hypertrophic cartilage. Hence, angiogenesis is dependent on Fe and the anti-angiogenic effects observed with CPX2 are not surprising, particularly because this chelator is more effective than DFO at preventing DNA synthesis in endothelial cells.2 Therefore, the anti-tumor activity of Fe chelators observed in vitro, in animal models and also in clinical trials,5, 6 could be due not only to the effect of chelators on tumor cells but also on the vital angiogenesis process. Another hypoxia-inducible gene upregulated by Fe chelation is erythropoietin (EPO).17 The expression of this molecule normally results in the increased production of erythrocytes from bone marrow during periods of hypoxia.17 In terms of cancer therapy, the use of EPO with radiation or chemotherapy was thought to alleviate fatigue during treatment.18 However, recent studies show that several breast, prostate and ovarian cancers express EPO receptors that can induce growth in the presence of EPO.18 Indeed, the use of synthetic EPO in combination with traditional chemotherapy or radiotherapy has contributed to poor patient prognosis in clinical trials.18 Although there has been no studies examining the growth of tumors in the presence of elevated EPO caused by Fe-deprivation, clinical trials using potent Fe chelators demonstrate that effective Fe-depletion reduces tumour growth.5, 7, 8 Thus, similarly to the case outlined above for VEGF-1, the anti-proliferative effects of potent Fe chelators may overwhelm EPO-mediated proliferation of these tumors. In conclusion, we suggest that despite the fact that chelators up-regulate the expression of the angiogenesis factor, VEGF-1 and erythrocyte proliferation factor, EPO, there is strong evidence to show that potent Fe chelators induce counteracting pathways involved in cell cycle arrest and apoptosis. Clearly, the extent of their anti-proliferative effects is determined by the ability of chelators to inhibit vital Fe-containing enzymes (e.g., ribonucleotide reductase) that are required for DNA synthesis and cell cycle progression. Under these conditions, the potential angiogenic capacity of Fe-depletion is overcome, resulting in inhibition of tumor growth.5, 6, 7, 8, 9, 10, 11, 12, 13, 14 Therefore, it may be possible to pharmacologically modulate the extent of Fe chelation to induce the required biological effect. For instance, chelators with poor membrane permeability that stimulate angiogenesis by weakly binding intracellular Fe pools may have therapeutic uses such as the healing of chronic wounds. In contrast, highly membrane permeable chelators that target Fe-dependent functions such as DNA synthesis could be potent anti-tumor agents.5, 6 Yours sincerely, Nghia T.V. Le, Des R. Richardson