Treatment of dilated cardiomyopathy in a mouse model of Friedreich's ataxia using N-acetylcysteine and identification of alterations in microRNA expression that could be involved in its pathogenesis.

Deficient expression of the mitochondrial protein, frataxin, leads to a deadly cardiomyopathy. Our laboratory reported the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation after frataxin deletion in the heart. This was due, in part, to a pronounced increase in Keap1. To assess if this can be therapeutically targeted, cells were incubated with N-acetylcysteine (NAC), or buthionine sulfoximine (BSO), which increases or decreases glutathione (GSH), respectively, or the NRF2-inducer, sulforaphane (SFN). While SFN significantly (p < 0.05) induced NRF2, KEAP1 and BACH1, NAC attenuated SFN-induced NRF2, KEAP1 and BACH1. The down-regulation of KEAP1 by NAC was of interest, as Keap1 is markedly increased in the MCK conditional frataxin knockout (MCK KO) mouse model and this could lead to the decreased Nrf2 levels. Considering this, MCK KO mice were treated with i.p. NAC (500- or 1500-mg/kg, 5 days/week for 5-weeks) and demonstrated slightly less (p > 0.05) body weight loss versus the vehicle-treated KO. However, NAC did not rescue the cardiomyopathy. To additionally examine the dys-regulation of Nrf2 upon frataxin deletion, studies assessed the role of microRNA (miRNA) in this process. In MCK KO mice, miR-144 was up-regulated, which down-regulates Nrf2. Furthermore, miRNA screening in MCK KO mice demonstrated 23 miRNAs from 756 screened were significantly (p < 0.05) altered in KOs versus WT littermates. Of these, miR-21*, miR-34c*, and miR-200c, demonstrated marked alterations, with functional clustering analysis showing they regulate genes linked to cardiac hypertrophy, cardiomyopathy, and oxidative stress, respectively.

Regulation and control of nitric oxide (NO) in macrophages: Protecting the "professional killer cell" from its own cytotoxic arsenal via MRP1 and GSTP1.

We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transports NO as dinitrosyl-dithiol-iron complexes (DNICs) composed of iron, NO and glutathione (GSH). Hence, this gas with contrasting anti- and pro-tumor effects, which has been assumed to be freely diffusible, is a tightly-regulated species in M1-MØs. These control systems prevent NO cytotoxicity and may be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets.

Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease.

Iron is a crucial transition metal for virtually all life. Two major destinations of iron within mammalian cells are the cytosolic iron-storage protein, ferritin, and mitochondria. In mitochondria, iron is utilized in critical anabolic pathways, including: iron-storage in mitochondrial ferritin, heme synthesis, and iron-sulfur cluster (ISC) biogenesis. Although the pathways involved in ISC synthesis in the mitochondria and cytosol have begun to be characterized, many crucial details remain unknown. In this review, we discuss major aspects of the journey of iron from its initial cellular uptake, its modes of trafficking within cells, to an overview of its downstream utilization in the cytoplasm and within mitochondria. The understanding of mitochondrial iron processing and its communication with other organelles/subcellular locations, such as the cytosol, has been elucidated by the analysis of certain diseases e.g., Friedreich's ataxia. Increased knowledge of the molecules and their mechanisms of action in iron processing pathways (e.g., ISC biogenesis) will shape the investigation of iron metabolism in human health and disease.

Dp44mT targets the AKT, TGF-ß and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells.

BACKGROUND: Effective treatment of prostate cancer should be based on targeting interactions between tumour cell signalling pathways and key converging downstream effectors. Here, we determined how the tumourigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumour-suppressive phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and transforming growth factor-ß (TGF-ß) pathways are integrated via the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumour agent, Dp44mT, may target these integrated pathways by increasing NDRG1 expression. METHODS: Protein expression in Dp44mT-treated normal human prostate epithelial cells and prostate cancer cells (PC-3, DU145) was assessed by western blotting. The role of NDRG1 was examined by transfection using an NDRG1 overexpression vector or shRNA. RESULTS: Dp44mT increased levels of tumour-suppressive PTEN, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras/MAPK signalling. Importantly, the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumour selectivity of these agents. Silencing NDRG1 expression increased phosphorylation of tumourigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, whereas NDRG1 overexpression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels. CONCLUSION: NDRG1 has an important role in mediating the tumour-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-ß and ERK pathways.

Thiosemicarbazones reprogram pancreatic cancer bidirectional oncogenic signaling between cancer cells and stellate cells to suppress desmoplasia.

Standard treatments have shown dismal activity against pancreatic cancer (PC), due in part to the development of a dense stroma (desmoplasia). This perspective discusses the development of the di-2-pyridylketone thiosemicarbazones that overcomes bidirectional oncogenic signaling between PC cells and pancreatic stellate cells (PSCs), which is critical for desmoplasia development. This activity is induced by the up-regulation of the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), which inhibits oncogenic signaling via HGF, IGF-1 and Sonic Hedgehog pathway. More recent studies have deciphered additional pathways including those mediated by Wnt and tenascin C that are secreted by PSCs to activate ß-catenin and YAP/TAZ signaling in PC cells. Suppression of bidirectional signaling between cell types presents a unique therapeutic opportunity.

Ascorbate-and iron-driven redox activity of Dp44mT and Emodin facilitates peroxidation of micelles and bicelles.

BACKGROUND: Iron (Fe)-induced oxidative stress leads to reactive oxygen species that damage biomembranes, with this mechanism being involved in the activity of some anti-cancer chemotherapeutics. METHODS: Herein, we compared the effect of the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), or the potential ligand, Emodin, on Fe-catalyzed lipid peroxidation in cell membrane models (micelles and bicelles). These studies were performed in the presence of hydrogen peroxide (H2O2) and the absence or presence of ascorbate. RESULTS: In the absence of ascorbate, Fe(II)/Emodin mixtures incubated with H2O2 demonstrated slight pro-oxidant properties on micelles versus Fe(II) alone, while the Fe(III)-Dp44mT complex exhibited marked antioxidant properties. Examining more physiologically relevant phospholipid-containing bicelles, the Fe(II)- and Fe(III)-Dp44mT complexes demonstrated antioxidant activity without ascorbate. Upon adding ascorbate, there was a significant increase in the peroxidation of micelles and bicelles in the presence of unchelated Fe(II) and H2O2. The addition of ascorbate to Fe(III)-Dp44mT substantially increased the peroxidation of micelles and bicelles, with the Fe(III)-Dp44mT complex being reduced by ascorbate to the Fe(II) state, explaining the increased reactivity. Electron paramagnetic resonance spectroscopy demonstrated ascorbyl radical anion generation after mixing ascorbate and Emodin, with signal intensity being enhanced by H2O2. This finding suggested Emodin semiquinone radical formation that could play a role in its reactivity via ascorbate-driven redox cycling. Examining cultured melanoma cells in vitro, ascorbate at pharmacological levels enhanced the anti-proliferative activity of Dp44mT and Emodin. CONCLUSIONS AND GENERAL SIGNIFICANCE: Ascorbate-driven redox cycling of Dp44mT and Emodin promotes their anti-proliferative activity.

Parkinson's disease: Alterations in iron and redox biology as a key to unlock therapeutic strategies.

A plethora of studies indicate that iron metabolism is dysregulated in Parkinson's disease (PD). The literature reveals well-documented alterations consistent with established dogma, but also intriguing paradoxical observations requiring mechanistic dissection. An important fact is the iron loading in dopaminergic neurons of the substantia nigra pars compacta (SNpc), which are the cells primarily affected in PD. Assessment of these changes reveal increased expression of proteins critical for iron uptake, namely transferrin receptor 1 and the divalent metal transporter 1 (DMT1), and decreased expression of the iron exporter, ferroportin-1 (FPN1). Consistent with this is the activation of iron regulator protein (IRP) RNA-binding activity, which is an important regulator of iron homeostasis, with its activation indicating cytosolic iron deficiency. In fact, IRPs bind to iron-responsive elements (IREs) in the 3ꞌ untranslated region (UTR) of certain mRNAs to stabilize their half-life, while binding to the 5ꞌ UTR prevents translation. Iron loading of dopaminergic neurons in PD may occur through these mechanisms, leading to increased neuronal iron and iron-mediated reactive oxygen species (ROS) generation. The "gold standard" histological marker of PD, Lewy bodies, are mainly composed of α-synuclein, the expression of which is markedly increased in PD. Of note, an atypical IRE exists in the α-synuclein 5ꞌ UTR that may explain its up-regulation by increased iron. This dysregulation could be impacted by the unique autonomous pacemaking of dopaminergic neurons of the SNpc that engages L-type Ca+2 channels, which imparts a bioenergetic energy deficit and mitochondrial redox stress. This dysfunction could then drive alterations in iron trafficking that attempt to rescue energy deficits such as the increased iron uptake to provide iron for key electron transport proteins. Considering the increased iron-loading in PD brains, therapies utilizing limited iron chelation have shown success. Greater therapeutic advancements should be possible once the exact molecular pathways of iron processing are dissected.

Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents.

Cancer contributes to 50% of deaths worldwide and new anti-tumour therapeutics with novel mechanisms of actions are essential to develop. Metabolic inhibitors represent an important class of anti-tumour agents and for many years, agents targeting the nutrient folate were developed for the treatment of cancer. This is because of the critical need of this factor for DNA synthesis. Similarly to folate, Fe is an essential cellular nutrient that is critical for DNA synthesis. However, in contrast to folate, there has been limited effort applied to specifically design and develop Fe chelators for the treatment of cancer. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) group of ligands that demonstrate marked and selective anti-tumour activity in vitro and also in vivo against a wide spectrum of tumours. Indeed, administration of these compounds to mice did not induce whole body Fe-depletion or disturbances in haematological or biochemical indices due to the very low doses required. The mechanism of action of these ligands includes alterations in expression of molecules involved in cell cycle control and metastasis suppression, as well as the generation of redox-active Fe complexes. This review examines the alterations in Fe metabolism in tumour cells and the systematic development of novel aroylhydrazone and thiosemicarbazone Fe chelators for cancer treatment.

The anti-tumor agent, Dp44mT, promotes nuclear translocation of TFEB via inhibition of the AMPK-mTORC1 axis.

Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and its analogues are potent anti-cancer agents through their ability to target lysosomes. Considering this, it was important to understand the mechanisms involved in the Dp44mT-mediated induction of autophagy and the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) as a critical autophagic regulator. As such, this investigation examined AMPK's role in the regulation of the transcription factor EB (TFEB), which transcribes genes involved in autophagy and lysosome biosynthesis. For the first time, this study demonstrated that Dp44mT induces translocation of TFEB to the nucleus. Furthermore, Dp44mT-mediated nuclear translocation of TFEB was AMPK-dependent. Considering that: (1) the mammalian target of rapamycin complex 1 (mTORC1) plays an important role in the regulation of TFEB; and (2) that AMPK is a known regulator of mTORC1, this study also elucidated the mechanisms through which Dp44mT regulates nuclear translocation of TFEB via AMPK. Silencing AMPK led to increased mTOR phosphorylation, that activates mTORC1. Since Dp44mT inhibits mTORC1 in an AMPK-dependent manner through raptor phosphorylation, Dp44mT is demonstrated to regulate TFEB translocation through dual mechanisms: AMPK activation, which inhibits mTOR, and inhibition of mTORC1 via phosphorylation of raptor. Collectively, Dp44mT-mediated activation of AMPK plays a crucial role in lysosomal biogenesis and TFEB function. As Dp44mT potently chelates copper and iron that are crucial for tumor growth, these studies provide insight into the regulatory mechanisms involved in intracellular clearance and energy metabolism that occur upon alterations in metal ion homeostasis.

Antioxidant defense mechanisms and its dysfunctional regulation in the mitochondrial disease, Friedreich's ataxia.

Redox stress is associated with the pathogenesis of a wide variety of disease states. This can be amplified potentially through redox active iron deposits in oxidatively active organelles such as the mitochondrion. There are a number of disease states, including Friedreich's ataxia (FA) and sideroblastic anemia, where iron metabolism is dysregulated and leads to mitochondrial iron accumulation. Considering FA, which is due to the decreased expression of the mitochondrial protein, frataxin, this iron accumulation does not occur within protective storage proteins such as mitochondrial ferritin. Instead, it forms unbound biomineral aggregates composed of high spin iron(III), phosphorous and sulfur, which probably contributes to the observed redox stress. There is also a dysregulated response to the ensuing redox assault, as the master regulator of oxidative stress, nuclear factor erythroid 2-related factor-2 (Nrf2), demonstrates marked down-regulation. The dysfunctional response of Nrf2 in FA is due to multiple mechanisms including: (1) up-regulation of Keap1 that is involved in Nrf2 degradation; (2) activation of the nuclear Nrf2 export/degradation machinery via glycogen synthase kinase-3ß (Gsk3ß) signaling; and (3) inhibited nuclear translocation of Nrf2. More recently, increased microRNA (miRNA) 144 expression has been demonstrated to down-regulate Nrf2 in several disease states, including an animal model of FA. Other miRNAs have also demonstrated to be dysregulated upon frataxin depletion in vivo in humans and animal models of FA. Collectively, frataxin depletion results in multiple, complex responses that lead to detrimental redox effects that could contribute to the mechanisms involved in the pathogenesis of FA.

Regulation of autophagy and apoptosis by Dp44mT-mediated activation of AMPK in pancreatic cancer cells.

Upon activation, the 5'-adenosine monophosphate-activated protein kinase (AMPK) increases catabolism, while inhibiting anabolism. The anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), activates AMPK in multiple tumor cell-types (Biochim. Biophys Acta 2016;1863:2916-2933). This acts as an initial cell "rescue response" after iron-depletion mediated by Dp44mT. Considering Dp44mT-mediated AMPK activation, the role of AMPK on Dp44mT cytotoxicity was examined. Dp44mT increased the p-AMPK/AMPK ratio in multiple tumor cell-types over short (24 h) and longer (72 h) incubations. Notably, Dp44mT was more effective in inhibiting tumor cell proliferation after AMPK silencing, potentially due to the loss of AMPK-mediated metabolic plasticity that protects cells against Dp44mT cytotoxicity. The silencing of AMPK-increased cellular cholesterol and stabilized lysosomes against Dp44mT-mediated lysosomal membrane permeabilization. This was substantiated by studies demonstrating that the cholesterol-depleting agent, methyl-ß-cyclodextrin (MßCD), restores Dp44mT-mediated lysosomal membrane permeabilization in AMPK silenced cells. The increased levels of cholesterol after AMPK silencing were independent of the ability of AMPK to inhibit the rate-limiting step of cholesterol synthesis via the inactivating phosphorylation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) at Ser872. In fact, Dp44mT did not increase phosphorylation of HMGCR at (Ser872), but decreased total HMGCR expression similarly in both the presence or absence of AMPK silencing. Dp44mT was demonstrated to increase autophagic initiation after AMPK silencing via an AMPK- and Beclin-1-independent mechanism. Further, there was increased cleaved caspase 3 and cleaved PARP after incubation of AMPK silenced cells with Dp44mT. Overall, AMPK silencing promotes Dp44mT anti-proliferative activity, suggesting a role for AMPK in rescuing its cytotoxicity by inhibiting autophagy and also apoptosis.

During mitosis ZEB1 "switches" from being a chromatin-bound epithelial gene repressor, to become a microtubule-associated protein.

Microtubules are polymers of α/ß-tubulin, with microtubule organization being regulated by microtubule-associated proteins (MAPs). Herein, we describe a novel role for the epithelial gene repressor, zinc finger E-box-binding homeobox 1 (ZEB1), that "switches" from a chromatin-associated protein during interphase, to a MAP that associates with α-, ß- and γ-tubulin during mitosis. Additionally, ZEB1 was also demonstrated to associate with γ-tubulin at the microtubule organizing center (MTOC). Using confocal microscopy, ZEB1 localization was predominantly nuclear during interphase, with α/ß-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during the stages of mitosis, ZEB1 co-localization with α-, ß-, and γ-tubulin was significantly increased, with the association commonly peaking during metaphase in multiple tumor cell-types. ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The increased interaction between ZEB1 and α-tubulin during mitosis was also confirmed using the proximity ligation assay. In contrast to ZEB1, its paralog ZEB2, was mainly perinuclear and cytoplasmic during interphase, showing some co-localization with α-tubulin during mitosis. Considering the association between ZEB1 with α/ß/γ-tubulin during mitosis, studies investigated ZEB1's role in the cell cycle. Silencing ZEB1 resulted in a G2-M arrest, which could be mediated by the up-regulation of p21Waf1/Cip1 and p27Kip1 that are known downstream targets repressed by ZEB1. However, it cannot be excluded the G2/M arrest observed after ZEB1 silencing is not due to its roles as a MAP. Collectively, ZEB1 plays a role as a MAP during mitosis and could be functionally involved in this process.

ATP7A is a novel target of retinoic acid receptor beta2 in neuroblastoma cells.

Increased retinoic acid receptor beta (RARbeta(2)) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARbeta(2) expression is a common feature of many human cancers, suggesting that RARbeta(2) may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARbeta(2) expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARbeta(2) protein alone was sufficient for the growth inhibitory effects of RARbeta(2) on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARbeta(2). The ectopic overexpression of the RARbeta(2) ABC domain was sufficient to induce ATP7A expression, whereas, RARbeta(2) siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor beta and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism.

The metastasis suppressor, NDRG1, differentially modulates the endoplasmic reticulum stress response.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), is a stress response protein that is involved in the inhibition of multiple oncogenic signaling pathways. Initial studies have linked NDRG1 and the endoplasmic reticulum (ER) stress response. Considering this, we extensively examined the mechanism by which NDRG1 regulates the ER stress response in pancreatic and colon cancer cells. We also examined the anti-cancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), which induces NDRG1 expression and causes ER stress. The expression of NDRG1 was demonstrated to regulate the three main arms of the ER stress response by: (1) increasing the expression of three major ER chaperones, binding immunoglobulin protein (BiP), calreticulin, and calnexin; (2) suppressing the protein kinase, RNA-activated (PKR)-like ER kinase (PERK); (3) inhibiting the inositol-requiring kinase 1α (IRE1α) arm; and (4) increasing the cleavage of activating transcription factor 6 (ATF6). An important finding was that NDRG1 enhances the anti-proliferative and anti-migratory activity of Dp44mT. This increased efficacy could be related to the following effects in the presence of Dp44mT and NDRG1, namely: markedly increased activation of the PERK target, eukaryotic translation initiation factor 2α (eIF2α); the maintenance of activating transcription factor 4 (ATF4) expression; high cytosolic Ca+2 that increases the sensitivity of cells to apoptosis via activation of the calmodulin-dependent kinase II (CaMKII) signaling cascade; and increased pro-apoptotic C/EBP-homologous protein (CHOP) expression. Collectively, this investigation dissects the molecular mechanisms through which NDRG1 manipulates the ER stress response and its ability to potentiate the activity of the potent anti-cancer agent, Dp44mT.

The melanoma tumor antigen, melanotransferrin (p97): a 25-year hallmark--from iron metabolism to tumorigenesis.

Melanotransferrin (MTf) or melanoma tumor antigen p97 is a transferrin (Tf) homolog that is found predominantly bound to the cell membrane via a glycosyl phosphatidylinositol anchor. The molecule is a member of the Tf superfamily and binds iron through a single high-affinity iron(III)-binding site. Since its discovery on the plasma membrane of melanoma cells, the function of MTf has remained intriguing, particularly in relation to its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways, e.g., DNA synthesis, it was important to understand the function of MTf in the transport of this vital nutrient. MTf has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis and cell migration. However, recent studies using a knockout mouse and post-transcriptional gene silencing have demonstrated that MTf is not involved in iron metabolism, but plays a vital role in melanoma cell proliferation and tumorigenesis. In this review, we discuss the possible biological functions of MTf, particularly in relation to cancer.

Iron chelation by clinically relevant anthracyclines: alteration in expression of iron-regulated genes and atypical changes in intracellular iron distribution and trafficking.

Anthracyclines are effective anticancer agents. However, their use is limited by cardiotoxicity, an effect linked to their ability to chelate iron and to perturb iron metabolism (Mol Pharmacol 68:261-271, 2005). These effects on iron-trafficking remain poorly understood, but they are important to decipher because treatment for anthracycline cardiotoxicity uses the chelator, dexrazoxane. Incubation of cells with doxorubicin (DOX) up-regulated mRNA levels of the iron-regulated genes transferrin receptor-1 (TfR1) and N-myc downstream-regulated gene-1 (Ndrg1). This effect was mediated by iron depletion, because it was reversed by adding iron and it was prevented by saturating the anthracycline metal binding site with iron. However, DOX did not act like a typical chelator, because it did not induce cellular iron mobilization. In the presence of DOX and (59)Fe-transferrin, iron-trafficking studies demonstrated ferritin-(59)Fe accumulation and decreased cytosolic-(59)Fe incorporation. This could induce cytosolic iron deficiency and increase TfR1 and Ndrg1 mRNA. Up-regulation of TfR1 and Ndrg1 by DOX was independent of anthracycline-mediated radical generation and occurred via hypoxia-inducible factor-1alpha-independent mechanisms. Despite increased TfR1 and Ndrg1 mRNA after DOX treatment, this agent decreased TfR1 and Ndrg1 protein expression. Hence, the effects of DOX on iron metabolism were complex because of its multiple effector mechanisms.

Protection against hydrogen peroxide-mediated cytotoxicity in Friedreich's ataxia fibroblasts using novel iron chelators of the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone class.

Iron-loading diseases remain an important problem because of the toxicity of iron-catalyzed redox reactions. Iron loading occurs in the mitochondria of Friedreich's ataxia (FA) patients and may play a role in its pathogenesis. This suggests that iron chelation therapy could be useful. We developed previously the lipophilic iron chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) ligands and identified 2-pyridylcarboxaldehyde 2-thiophenecarboxyl hydrazone (PCTH) as the most promising analog. Hence, this study assessed the efficacy of PCTH and other PCIH analogs compared with various chelators, including deferiprone and desferrioxamine (DFO). Age- and sex-matched control and FA fibroblasts were preincubated with iron chelators and subsequently challenged with 50 microM H2O2 for up to 24 h. The current study demonstrates an interesting structure-activity relationship among the closely related PCIH series of ligands, with only PCTH being highly effective at preventing H2O2-induced cytotoxicity. PCTH increased FA fibroblast cell viability by up to 70%, whereas DFO rescued viability by 1 to 5% only. Hence, PCTH, which was well tolerated by cells was far more effective than DFO at preventing oxidative stress. It is noteworthy that kinetic studies demonstrated PCTH to rapidly penetrate cells to induce 59Fe efflux, whereas DFO, PCIH, 2-pyridylcarboxaldehyde benzoyl hydrazone, and 2-pyridylcarboxaldehyde m-bromobenzoyl hydrazone were far slower, indicating it is the rate of chelator permeation that is crucial for protection against H2O2. In addition, PCTH was found to be as effective as or more effective than conventional radical scavengers or the antioxidant idebenone (which has undergone clinical trials) at protecting cells against H2O2-mediated cytotoxicity. These findings further indicate the potential of PCTH for treatment of iron overload.

Identification of distinct changes in gene expression after modulation of melanoma tumor antigen p97 (melanotransferrin) in multiple models in vitro and in vivo.

Melanoma tumor antigen p97 or melanotransferrin (MTf) is an iron (Fe)-binding protein with high homology to serum transferrin. MTf is expressed at very low levels in normal tissues and in high amounts in melanoma cells although its function remains elusive. To understand the function of MTf, we utilized whole-genome microarray analysis to examine the gene expression profile of five models after modulating MTf expression. These models included two new stably transfected MTf hyper-expression models (SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type (SK-Mel-28 melanoma) where MTf was down-regulated by post-transcriptional gene silencing. These findings were compared with alterations in gene expression identified using the MTf-/- mice. In addition, the changes identified from the microarray data were also assessed in a new model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, whereas MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and up-regulation, we identified three genes modulated by MTf. These included ATP-binding cassette subfamily B member 5, whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase and transcription factor 4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. This study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation.

Examination of the distribution of the transferrin homologue, melanotransferrin (tumour antigen p97), in mouse and human.

Melanotransferrin (MTf) is a transferrin homologue initially identified in melanoma cells. Serum transferrin (Tf) contains two iron (Fe)-binding sites and plays a vital role in Fe transport. However, human MTf has only a single, high affinity, Fe-binding site. Furthermore, while isolated MTf can bind Fe, it plays little role in Fe uptake by cells and its function remains elusive. To further understand the biological role of this molecule, we examined the expression profile of mouse MTf (mMTf) and human MTf (hMTf) and the splice variant of the latter. Analysis of mMTf in 18 normal mouse tissues and 4 embryonic stages (7-17 days) using an RNA dot blot demonstrated it was expressed at high levels in the pancreas, salivary gland and epididymis of the adult, while embryonic tissues showed low expression. The expression pattern was very different from that of mouse transferrin receptor 1 (TfR1) mRNA, which was found at high levels in the spleen and embryo. Using the more sensitive RT-PCR technique, mMTf expression was demonstrated across all 24 normal mouse tissues assessed. Analysis of the mMTf genomic sequence predicted only one mMTf transcript, although two putative transcripts were found in the testis using Northern blotting. An alternate hMTf transcript, h delta MTf, has been identified by others, although its tissue distribution was not previously examined. In human heart and skeletal muscle, three putative hMTf transcripts were identified at approximately 2, 3 and 4 kb, the smallest transcript being consistent with h delta MTf. The two larger transcripts were also found in 10 other human tissues. The h delta MTf transcript was detected using RT-PCR and Southern blotting in tumour-derived cell lines, with the highest expression being identified in melanoma cells. Immunohistochemistry showed that hMTf was expressed primarily within epithelia. In fact, the most pronounced expression was within the epidermis of the skin, tubules of the kidney and the ducts of sweat and salivary glands. The distribution of MTf and its splice variants may provide clues to their possible biological roles.

Mobilization of iron from neoplastic cells by some iron chelators is an energy-dependent process.

Iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class may be useful agents to treat Fe overload disease and also cancer. These ligands possess high activity at mobilizing 59Fe from neoplastic cells, and the present study has been designed to examine whether their marked activity may be related to an energy-dependent transport process across the cell membrane. Initial experiments examined the release of 59Fe from SK-N-MC neuroblastoma (NB) cells prelabelled for 3 h at 37 degrees C with 59Fe-transferrin (1.25 microM) and then reincubated in the presence and absence of the chelators for 3 h at 4 degrees C or 37 degrees C. Prelabelled cells released 4-5% of total cellular 59Fe when reincubated in minimum essential medium at 4 degrees C or 37 degrees C. When the chelators desferrioxamine (DFO; 0.1 mM) or PIH (0.1 mM) were reincubated with labelled cells at 4 degrees C, they mobilized only 4-5% of cellular 59Fe, whereas as 37 degrees C, these ligands mobilized 21% and 48% of cell 59Fe, respectively. The lipophilic PIH analogue, 311 (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 0.1 mM), which exhibits high anti-proliferative activity, released 10% and 53% of cellular 59Fe when reincubated with prelabelled cells at 4 degrees C and 37 degrees C, respectively. Almost identical results were obtained using the SK-Mel-28 melanoma cell line. These data suggest that perhaps temperature-dependent mechanisms are essential for 59Fe mobilization from these cells. Interestingly, the metabolic inhibitors, 2,4-dinitrophenol, oligomycin, rotenone, and sodium azide, markedly decreased 59Fe mobilization mediated by PIH, but had either no effect or much less effect on 59Fe release by 311. Considering that an ATP-dependent process was involved in 59Fe release by PIH, further studies examined 4 widely used inhibitors of the multi-drug efflux pump P-glycoprotein (P-gp). All of these inhibitors, namely, verapamil (Ver), cyclosporin A (CsA), reserpine (Res) and quinine (Qui), decreased 59Fe mobilization by PIH but had little or no effect on 59Fe release mediated by analogue 311. Further, both CsA and Ver increased the proportion of ethanol-soluble 59Fe within cells in the presence of PIH, suggesting inhibited transport of the 59Fe complex from the cell. However, when PIH-mediated 59Fe release was compared between a well characterized Chinese hamster ovary cell line (CHRB30) expressing high levels of P-gp and the relevant control cell line (AuxB1), no appreciable difference in the kinetics of 59Fe release were found. In contrast, it was intriguing that the CHRB30 cells released more 59Fe into control medium (i.e., without PIH) than the AuxB1 control line (16.7% compared to 5.9%, respectively). In summary, the results suggest that a temperature- and energy-dependent process was involved in the efflux of the PIH-59Fe complex from the cells. In contrast, 59Fe release mediated by 311 was temperature-dependent but not energy-dependent, and could occur by simple diffusion or passive transport. Further studies investigating the membrane transport of Fe chelators may be useful in designing regimes that potentiate their anti-neoplastic effects.