The use of iron chelators as anti-proliferative agents against cancer: Molecular mechanisms behind the cell cycle arrest and apoptosis

Iron (Fe) is a fundamental requirement for life since it is involved in many cellular processes critical for growth and proliferation. Indeed, studies which deplete Fe from neoplastic cells using specific chelating agents result in G1/S arrest and apoptosis. However, the precise role of Fe in the control of the cell cycle remains unclear. Chapter one of this thesis, discusses the potential mechanism(s) by which Fe chelators cause cell cycle arrest and apoptosis. Recent studies have shown that this process involves multiple molecules and is highly complex. Thus, the studies presented in this thesis assess the effects of specific high affinity Fe chelators on the expression of molecules that may play important roles in cellular arrest and apoptosis. The general methodology used in these studies is summarized in chapter 2.;In chapter 4, gene arrays were used to assess the expression of molecules associated with cell cycle control and the p53 pathway following the incubation of cells with DFO, 311 or Act D. The N-myc downstream regulated gene 1 (Ndrg1) was the only molecule identified that was specifically up-regulated by Fe chelation and not DNA-damage. Although the exact function of the Ndrg1 protein is unclear, previous studies have shown that this molecule markedly slows down tumor growth (Kurdistani et al. Cancer Res. 1998:58:4439-44) and acts as a potent metastasis suppressor protein (Bandyopadhyay et al. Cancer Res. 2003:63,1731-6). Subsequent experiments examined the mRNA and protein expression of Ndrg1 using reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Incubation of cells with DFO or 311 resulted in a marked increase in the expression of Ndrg1 mRNA and protein within 3 h and 6 h, respectively. This increase in Ndrg1 after incubation with chelators was not found with their Fe complexes nor when the Fe-binding site had been synthetically inactivated. In addition, the induction of Ndrg1 following Fe chelation was correlated with the ligands' permeability and anti-proliferative activity. Indeed, the up-regulation of Ndrg1 by Fe chelators can be readily reversed by Fe-repletion. Collectively, this indicates that the induction of Ndrg1 was dependent on intracellular Fe levels. Previous studies demonstrated for the first time that the transactivation of Ndrg1 was dependent on HIF-1alpha and p53 stabilization. In contrast, investigations using fibroblasts derived from a HIF-1alpha knockout (HIF-1alpha-KO) mouse demonstrated that the transcriptional up-regulation of Ndrg1 following Fe chelation was mediated by a HIF-1alpha-dependent and -independent mechanism. In addition, subsequent experiments using the p53-deficient H1299 lung carcinoma cell line revealed that the transactivation of Ndrg1 was not dependent on p53 status after Fe-depletion. Collectively, the presented studies suggest that Ndrg1 may be a novel link between Fe metabolism and the control of proliferation. The up-regulation of Ndrg1 by potent Fe chelators is significant and may be beneficial in the prevention of tumor metastasis.;In conclusion, this thesis demonstrates that the molecular mechanisms behind the G1/S arrest and apoptosis involved multiple molecules following Fe chelation. Indeed, this study shows a potential mechanism by which Fe chelators may cause cell cycle dysregulation through the down-regulation of nuclear p21CIP1/WAF1 protein levels. In addition, the work presented identifies the up-regulation of the metastasis suppressor protein, Ndrg1, following Fe-deprivation. Collectively, the results indicate that potent Fe chelators can not only be used as anti-proliferative agents, but may also be beneficial at inhibiting tumor cell metastasis.;In previous investigations Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were shown to be far more potent anti-tumor agents than the clinically used ligand, desferrioxamine (DFO). Studies detailed in chapter 3, examined the expression of the tumor suppressor protein p53 and the universal cyclin-dependent kinase inhibitor p21CIP1/WAF1 following Fe-deprivation. To further characterize the effects of chelators on cell cycle arrest, experiments compared their activity to the DNA-damaging agents, actinomycin D (Act D) and cisplatin (CP). The latter two compounds increase the expression of p53 and its target gene p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased accumulation of nuclear p53, with the effect being more pronounced for Act D and CP. As expected, both Act D and CP markedly increased nuclear p21 CIP1/WAF1 protein levels, while DFO and 311 caused a significant (p < 0.0004) decrease. This result was surprising, since the WAF1 gene which encodes the universal cyclin dependent kinase inhibitor p21CIP1/WAF1 was markedly up-regulated at the mRNA level following Fe chelation. Proteasomal inhibition studies demonstrated the partial involvement of proteasomes in decreasing nuclear p21CIP1/WAF1 levels following DFO treatment. In contrast, the incubation of 311-treated cells with proteasomal inhibitors did not reverse the down-regulation of p21CIP1/WAF1 when compared to the control. Immunofluorescence experiments showed that only DNA-damaging agents and not Fe chelators increased the nuclear translocation of p21CIP1/WAF1. Collectively, this suggests that the chelators prevented translation of WAF1. Moreover, this decrease in nuclear p21CIP1/WAF1 protein expression did not appear to be due to a general effect in which Fe chelators inhibited mRNA translation since the transferrin receptor 1 was markedly up-regulated (15--21-fold) by DFO or 311. The combination of 311 with Act D or CP prevented the marked p21 CIP1/WAF1 nuclear accumulation normally observed in response to these DNA-damaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 expression was reversed by the Fe-donor, ferric ammonium citrate (FAC), indicating that WAF1 translation was dependent on intracellular Fe levels. This study is the first to demonstrate that Fe chelators markedly up-regulate the mRNA expression of WAF1 but paradoxically inhibit its translation. The down-regulation of p21CIP1/WAF1 protein by chelators may be a novel mechanism by which these ligands cause G1/S arrest through cell cycle dysregulation.