Iron Induces Cell Death and Strengthens the Efficacy of Antiandrogen Therapy in Prostate Cancer Models.

PURPOSE: In search of novel strategies to improve the outcome of advanced prostate cancer, we considered that prostate cancer cells rearrange iron homeostasis, favoring iron uptake and proliferation. We exploited this adaptation by exposing prostate cancer preclinical models to high-dose iron to induce toxicity and disrupt adaptation to androgen starvation. EXPERIMENTAL DESIGN: We analyzed markers of cell viability and mechanisms underlying iron toxicity in androgen receptor-positive VCaP and LNCaP, castration-resistant DU-145 and PC-3, and murine TRAMP-C2 cells treated with iron and/or the antiandrogen bicalutamide. We validated the results in vivo in VCaP and PC-3 xenografts and in TRAMP-C2 injected mice treated with iron and/or bicalutamide. RESULTS: Iron was toxic for all prostate cancer cells. In particular, VCaP, LNCaP, and TRAMP-C2 were highly iron sensitive. Toxicity was mediated by oxidative stress, which primarily affected lipids, promoting ferroptosis. In highly sensitive cells, iron additionally caused protein damage. High-basal iron content and oxidative status defined high iron sensitivity. Bicalutamide-iron combination exacerbated oxidative damage and cell death, triggering protein oxidation also in poorly iron-sensitive DU-145 and PC-3 cells.In vivo, iron reduced tumor growth in TRAMP-C2 and VCaP mice. In PC-3 xenografts, bicalutamide-iron combination caused protein oxidation and successfully impaired tumor expansion while single compounds were ineffective. Macrophages influenced body iron distribution but did not limit the iron effect on tumor expansion. CONCLUSIONS: Our models allow us to dissect the direct iron effect on cancer cells. We demonstrate the proof of principle that iron toxicity inhibits prostate cancer cell proliferation, proposing a novel tool to strengthen antiandrogen treatment efficacy.

Coenzyme A corrects pathological defects in human neurons of PANK2-associated neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is an early onset and severely disabling neurodegenerative disease for which no therapy is available. PKAN is caused by mutations in PANK2, which encodes for the mitochondrial enzyme pantothenate kinase 2. Its function is to catalyze the first limiting step of Coenzyme A (CoA) biosynthesis. We generated induced pluripotent stem cells from PKAN patients and showed that their derived neurons exhibited premature death, increased ROS production, mitochondrial dysfunctions-including impairment of mitochondrial iron-dependent biosynthesis-and major membrane excitability defects. CoA supplementation prevented neuronal death and ROS formation by restoring mitochondrial and neuronal functionality. Our findings provide direct evidence that PANK2 malfunctioning is responsible for abnormal phenotypes in human neuronal cells and indicate CoA treatment as a possible therapeutic intervention.

Serum hepcidin levels and muscle iron proteins in humans injected with low- or high-dose erythropoietin.

Inhibition of hepcidin expression by erythropoietic signals is of great physiological importance; however, the inhibitory pathways remain poorly understood. To investigate (i) the direct effect of erythropoietin (Epo) and (ii) the contribution of putative mediators on hepcidin repression, healthy volunteers were injected with a single dose of Epo, either low (63 IU/kg, n = 8) or high (400 IU/kg, n = 6). Low-dose Epo provoked hepcidin down-modulation within 24 h; the effect was not immediate as hepcidin circadian variations were still present following injection. High-dose Epo induced no additional effect on the hepcidin response, that is hepcidin diurnal fluctuations were not abolished in spite of extremely high Epo levels. We did not find significant changes in putative mediators of hepcidin repression, such as transferrin saturation, soluble transferrin receptor, or growth differentiation factor 15. Furthermore, the potential hepcidin inhibitor, soluble hemojuvelin, was found unaltered by Epo stimulation. This finding was consistent with the absence of signs of iron deficiency observed at the level of skeletal muscle tissue. Our data suggest that hepcidin repression by erythropoietic signals in humans may not be controlled directly by Epo, but mediated by a still undefined factor.

Iron increases the susceptibility of multiple myeloma cells to bortezomib.

Multiple myeloma is a malignant still incurable plasma cell disorder. Pharmacological treatment based on proteasome inhibition has improved patient outcome; however, bortezomib-resistance remains a major clinical problem. Inhibition of proteasome functionality affects cellular iron homeostasis and iron is a potent inducer of reactive oxygen species and cell death, unless safely stored in ferritin. We explored the potential role of iron in bortezomib-resistance. We analyzed iron proteins, oxidative status and cell viability in 7 multiple myeloma cell lines and in plasma cells from 5 patients. Cells were treated with increasing bortezomib concentrations with or without iron supplementation. We reduced ferritin levels by both shRNA technology and by drug-induced iron starvation. Multiple myeloma cell lines are characterized by distinct ferritin levels, which directly correlate with bortezomib resistance. We observed that iron supplementation upon bortezomib promotes protein oxidation and cell death, and that iron toxicity inversely correlates with basal ferritin levels. Bortezomib prevents ferritin upregulation in response to iron, thus limiting the ability to buffer reactive oxygen species. Consequently, reduction of basal ferritin levels increases both bortezomib sensitivity and iron toxicity. In patients' cells, we confirmed that bortezomib prevents ferritin increase, that iron supplementation upon bortezomib increases cell death and that ferritin reduction overcomes bortezomib resistance. Bortezomib affects iron homeostasis, sensitizing cells to oxidative damage. Modulation of iron status is a strategy worth exploring to improve the efficacy of proteasome inhibition therapies.

Skin fibroblasts from pantothenate kinase-associated neurodegeneration patients show altered cellular oxidative status and have defective iron-handling properties.

Pantothenate kinase-associated neurodegeneration (PKAN) is a neurodegenerative disease belonging to the group of neurodegeneration with brain iron accumulation disorders. It is characterized by progressive impairments in movement, speech and cognition. The disease is inherited in a recessive manner due to mutations in the Pantothenate Kinase-2 (PANK2) gene that encodes a mitochondrial protein involved in Coenzyme A synthesis. To investigate the link between a PANK2 gene defect and iron accumulation, we analyzed primary skin fibroblasts from three PKAN patients and three unaffected subjects. The oxidative status of the cells and their ability to respond to iron were analyzed in both basal and iron supplementation conditions. In basal conditions, PKAN fibroblasts show an increase in carbonylated proteins and altered expression of antioxidant enzymes with respect to the controls. After iron supplementation, the PKAN fibroblasts had a defective response to the additional iron. Under these conditions, ferritins were up-regulated and Transferrin Receptor 1 (TfR1) was down-regulated to a minor extent in patients compared with the controls. Analysis of iron regulatory proteins (IRPs) reveals that, with respect to the controls, PKAN fibroblasts have a reduced amount of membrane-associated mRNA-bound IRP1, which responds imperfectly to iron. This accounts for the defective expression of ferritin and TfR1 in patients' cells. The inaccurate quantity of these proteins produced a higher bioactive labile iron pool and consequently increased iron-dependent reactive oxygen species formation. Our results suggest that Pank2 deficiency promotes an increased oxidative status that is further enhanced by the addition of iron, potentially causing damage in cells.

Loss of the von Hippel Lindau tumor suppressor disrupts iron homeostasis in renal carcinoma cells.

Given the modulation of iron metabolism by hypoxia and the high iron requirement of neoplastic cells, we investigated iron metabolism in a human renal cancer cell line with a mutated von Hippel Lindau (VHL) tumor suppressor gene (RCC10) and in a transfectant clone with wild-type VHL (RCC63). The loss of VHL strongly up-regulated transferrin receptor expression in RCC10 cells as a result of hypoxia inducible factor-1 (HIF-1)-mediated transcriptional activation, leading to an increased uptake of transferrin-bound 55Fe. Increased iron availability did not compromise the resistance of VHL-defective cells to oxidative stress or promote faster cell multiplication. Surprisingly, the content of ferritin H and L subunits and ferritin mRNA levels were considerably lower in the RCC10 than in the RCC63 cells. Despite the similarities between HIF-1 and iron regulatory protein 2 (IRP2), we found no evidence of specific regulation of IRP2 by VHL. However, both IRP2 and IRP1 were slightly activated in RCC10 cells, thus indicating that this cell line has a somewhat reduced labile iron pool (LIP). The finding that RCC10 cells had a lower ferritin content but more ferritin-associated 55Fe than RCC63 explains why VHL-lacking cells may have a smaller LIP despite increased iron uptake. We also found a correlation between cytoprotection from iron-mediated damage and efficient incorporation into ferritin of both transferrin and non-transferrin-bound 55Fe. This study shows that, like oncogene activation, the loss of an oncosuppressor rearranges the expression pattern of the genes of iron metabolism to increase iron availability. However, in the case of VHL loss, mechanisms affecting iron handling by ferritin somehow counteract the effects that the reduced content of this protective protein may have on proliferation and oxidant sensitivity.

Ferroportin gene silencing induces iron retention and enhances ferritin synthesis in human macrophages.

Missense mutations in the ferroportin gene (SLC11A3) result in haemochromatosis type 4 [HFE4, Online Mendelian Inheritance in Man (OMIM) reference 606069] or ferroportin disease, an autosomal dominant disorder characterized by predominantly reticuloendothelial iron accumulation. To verify whether HFE4 is caused by defective iron recycling because of loss of functionality of ferroportin, we down-regulated SLC11A gene expression in human macrophages by using small interfering RNAs (siRNAs). Transfection experiments with ferroportin siRNAs resulted in a marked reduction (about two-thirds on average) in ferroportin mRNA levels as detected by quantitative real time polymerase chain reaction. When macrophages were grown in medium supplemented with iron, cells transfected with siRNAs displayed three- to eightfold increases in staining intensities following Perls reaction. These macrophages also showed significant increases in H-ferritin content. The observation that ferroportin mRNA down-regulation to levels compatible with haplo-insufficiency causes increased iron retention and H-ferritin synthesis in cultured macrophages has important implications. First, this indicates that ferroportin levels must be finely regulated in order to maintain cellular iron homeostasis, and that both copies of SLC11A3 must function efficiently to prevent iron accumulation. Second, this observation supports the hypothesis that reticuloendothelial iron overload in patients with ferroportin disease is caused by loss-of-function mutations in the SLC11A3 gene that mainly impair macrophage iron recycling.

Analysis of the biologic functions of H- and L-ferritins in HeLa cells by transfection with siRNAs and cDNAs: evidence for a proliferative role of L-ferritin.

We describe the use of small interfering RNAs (siRNAs) to down-regulate H- and L-ferritin levels in HeLa cells. siRNAs repressed H- and L-ferritin expression to about 20% to 25% of the background level in both stable and transient transfections. HeLa cells transfected with H- and L-ferritin cDNAs were analyzed in parallel to compare the effects of ferritin up- and down-regulation. We found that large modifications of L-ferritin levels did not affect iron availability in HeLa cells but positively affected cell proliferation rate in an iron-independent manner. The transient down-regulation of H-ferritin modified cellular iron availability and resistance to oxidative damage, as expected. In contrast, the stable suppression of H-ferritin in HeLa cell clones transfected with siRNAs did not increase cellular iron availability but made cells less resistant to iron supplementation and chelation. The results indicate that L-ferritin has no direct effects on cellular iron homeostasis in HeLa cells, while it has new, iron-unrelated functions. In addition, they suggest that H-ferritin function is to act as an iron buffer.

Role of iron and ferritin in TNFalpha-induced apoptosis in HeLa cells.

We found that tumor necrosis factor alpha (TNFalpha)-induced apoptosis in HeLa cells was accompanied by a approximately 2-fold increase in H- and L-ferritin and a decrease in transferrin receptor, two indices of increased iron availability. Iron supplementation and overexpression of H-ferritin or its mutant with an inactivated ferroxidase center reduced by about approximately 50% the number of apoptotic cells after TNFalpha-treatment, while overexpression of L-ferritin was ineffective. The data indicate that H-ferritin has an anti-apoptotic activity unrelated to its ferroxidase activity and to its capacity to modify cellular iron metabolism.