Meeting the Challenge 2: Identification of Potential Chemical Probes for Parkinson's Disease from Ligusticum chuanxiong Hort Using Cytological Profiling.

Traditional Chinese medicine (TCM) has been around for thousands of years and is increasingly gaining popularity in the Western world to treat various complex disorders including the incurable neurodegenerative condition, Parkinson's Disease (PD). One of the many directions in recent studies of PD is utilizing the phenotypic assay, or cytological profiling, to evaluate the phenotypic changes of PD-implicated cellular components in patient-derived olfactory neuroepithelial (hONS) cells, upon treating the cells with extracts or pure compounds. To obtain small molecules for studies utilizing PD phenotyping assays, Ligusticum chuanxiong Hort was selected for analysis as it is a popular Chinese herbal medicine used for treating PD-like symptoms. Fifty-three secondary metabolites, including six new compounds, were isolated from the ethanolic extract of L. chuanxiong; their structures were elucidated based on several spectroscopic techniques such as NMR, MS, Fourier transform infrared (FTIR), UV, and theoretical density functional theory (DFT) calculations. Cytological profiling of the afforded natural products against PD hONS cells revealed 34 compounds strongly perturbated the staining of several cellular organelles. In fact, greaterthan 1.5-fold change was observed compared to the control (dimethyl sulfoxide; DMSO), with early endosome, lysosome, and autophagosome (LC3b) being particularly affected. Given these biological compartments are closely related to PD pathogenesis, the results helped rationalize the traditional medicinal use of L. chuanxiong in PD treatment. Further, the hit compounds can serve as chemical probes to map the molecular pathways underlying PD, potentially leading to new therapeutic targets for PD.

Innovative therapies for neuroblastoma: The surprisingly potent role of iron chelation in up-regulating metastasis and tumor suppressors and down-regulating the key oncogene, N-myc.

Iron is an indispensable requirement for essential biological processes in cancer cells. Due to the greater proliferation of neoplastic cells, their demand for iron is considerably higher relative to normal cells, making them highly susceptible to iron depletion. Understanding this sensitive relationship led to research exploring the effect of iron chelation therapy for cancer treatment. The classical iron-binding ligand, desferrioxamine (DFO), has demonstrated effective anti-proliferative activity against many cancer-types, particularly neuroblastoma tumors, and has the surprising activity of down-regulating the potent oncogene, N-myc, which is a major oncogenic driver in neuroblastoma. Even more significant is the ability of DFO to simultaneously up-regulate the potent metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1), which plays a plethora of roles in suppressing a variety of oncogenic signaling pathways. However, DFO suffers the disadvantage of demonstrating poor membrane permeability and short plasma half-life, requiring administration by prolonged subcutaneous or intravenous infusions. Considering this, the specifically designed di-2-pyridylketone thiosemicarbazone (DpT) series of metal-binding ligands was developed in our laboratory. The lead agent from the first generation DpT series, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), showed exceptional anti-cancer properties compared to DFO. However, it exhibited cardiotoxicity in mouse models at higher dosages. Therefore, a second generation of agents was developed with the lead compound being di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) that progressed to Phase I clinical trials. Importantly, DpC showed better anti-proliferative activity than Dp44mT and no cardiotoxicity, demonstrating effective anti-cancer activity against neuroblastoma tumors in vivo.

The potential of the novel NAD+ supplementing agent, SNH6, as a therapeutic strategy for the treatment of Friedreich's ataxia.

Friedreich's ataxia (FA) is due to deficiency of the mitochondrial protein, frataxin, which results in multiple pathologies including a deadly, hypertrophic cardiomyopathy. Frataxin loss leads to deleterious accumulations of redox-active, mitochondrial iron, and suppressed mitochondrial bioenergetics. Hence, there is an urgent need to develop innovative pharmaceuticals. Herein, the activity of the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazone (SNH6), was assessed in vivo using the well-characterized muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse model of FA. The design of SNH6 incorporated a dual-mechanism mediating: (1) NAD+-supplementation to restore cardiac bioenergetics; and (2) iron chelation to remove toxic mitochondrial iron. In these studies, MCK wild-type (WT) and KO mice were treated for 4-weeks from the asymptomatic age of 4.5-weeks to 8.5-weeks of age, where the mouse displays an overt cardiomyopathy. SNH6-treatment significantly elevated NAD+ and markedly increased NAD+ consumption in WT and KO hearts. In SNH6-treated KO mice, nuclear Sirt1 activity was also significantly increased together with the NAD+-metabolic product, nicotinamide (NAM). Therefore, NAD+-supplementation by SNH6 aided mitochondrial function and cardiac bioenergetics. SNH6 also chelated iron in cultured cardiac cells and also removed iron-loading in vivo from the MCK KO heart. Despite its dual beneficial properties of supplementing NAD+ and chelating iron, SNH6 did not mitigate cardiomyopathy development in the MCK KO mouse. Collectively, SNH6 is an innovative therapeutic with marked pharmacological efficacy, which successfully enhanced cardiac NAD+ and nuclear Sirt1 activity and reduced cardiac iron-loading in MCK KO mice. No other pharmaceutical yet designed exhibits both these effective pharmacological properties.

Novel and potent anti-tumor and anti-metastatic di-2-pyridylketone thiosemicarbazones demonstrate marked differences in pharmacology between the first and second generation lead agents.

Di(2-pyridyl)ketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di(2-pyridyl)ketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) are novel, highly potent and selective anti-tumor and anti-metastatic drugs. Despite their structural similarity, these agents differ in their efficacy and toxicity in-vivo. Considering this, a comparison of their pharmacokinetic and pharmaco/toxico-dynamic properties was conducted to reveal if these factors are involved in their differential activity. Both compounds were administered to Wistar rats intravenously (2 mg/kg) and their metabolism and disposition were studied using UHPLC-MS/MS. The cytotoxicity of both thiosemicarbazones and their metabolites was also examined using MCF-7, HL-60 and HCT116 tumor cells and 3T3 fibroblasts and H9c2 cardiac myoblasts. Their intracellular iron-binding ability was characterized by the Calcein-AM assay and their iron mobilization efficacy was evaluated. In contrast to DpC, Dp44mT undergoes rapid demethylation in-vivo, which may be related to its markedly faster elimination (T1/2 = 1.7 h for Dp44mT vs. 10.7 h for DpC) and lower exposure. Incubation of these compounds with cancer cells or cardiac myoblasts did not result in any significant metabolism in-vitro. The metabolism of Dp44mT in-vivo resulted in decreased anti-cancer activity and toxicity. In conclusion, marked differences in the pharmacology of Dp44mT and DpC were observed and highlight the favorable pharmacokinetics of DpC for cancer treatment.

Adenosine monophosphate-activated kinase and its key role in catabolism: structure, regulation, biological activity, and pharmacological activation.

Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor, which once activated, plays a role in several processes within the cell to restore energy homeostasis. The protein enhances catabolic pathways, such as ß-oxidation and autophagy, to generate ATP, and inhibits anabolic processes that require energy, including fatty acid, cholesterol, and protein synthesis. Due to its key role in the regulation of critical cellular pathways, deregulation of AMPK is associated with the pathology of many diseases, including cancer, Wolff-Parkinson-White syndrome, neurodegenerative disorders, diabetes, and metabolic syndrome. In fact, AMPK is a target of some pharmacological agents implemented in the treatment of diabetes (metformin and thiazolidinediones) as well as other naturally derived products, such as berberine, which is used in traditional medicine. Due to its critical role in the cell and the pathology of several disorders, research into developing AMPK as a therapeutic target is becoming a burgeoning and exciting field of pharmacological research. A profound understanding of the regulation and activity of AMPK would enhance its development as a promising therapeutic target.

The anticancer agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes prosurvival autophagy by two mechanisms: persistent induction of autophagosome synthesis and impairment of lysosomal integrity.

Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death.

Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.

Cellular iron depletion and the mechanisms involved in the iron-dependent regulation of the growth arrest and DNA damage family of genes.

Iron plays a crucial part in proliferation while iron deficiency results in G(1)/S arrest, DNA damage, and apoptosis. However, the precise role of iron in cell cycle control remains unclear. We showed that iron depletion using the iron chelators, desferrioxamine (DFO), or 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311), increased the mRNA levels of the growth arrest and DNA damage 45α gene, GADD45α (Darnell, G. and Richardson, D. R. (1999) Blood 94, 781-792). In this study, we examined the effect of iron depletion on up-regulating GADD family members involved in growth control, including cell cycle arrest, apoptosis, and DNA repair, making them therapeutic targets for tumor suppression. We showed the GADD family members were up-regulated by cellular iron depletion. Further, up-regulation of GADD45α after iron deprivation was independent of hypoxia-inducible factor-1α (HIF-1α), octamer-1 (Oct-1), p53 and early growth response 1 (Egr1). We then analyzed the regulatory elements responsible for iron depletion-mediated regulation of GADD45α and identified the specific transcription factor/s involved. This region was within -117 bp and -81 bp relative to the start codon where the consensus sequences of three transcription factors are located: the CCAAT-binding factor/nuclear factor-Y (NF-Y), the stabilizing molecule v-MYB and the enhancer, CCAAT enhancer-binding protein (CEBPα). Mutation analysis, shRNA studies, Western blotting, and electrophoretic mobility shift assays led to the identification of NF-Y in the transcriptional up-regulation of GADD45α after iron depletion. Furthermore, like GADD45α, NF-YA was up-regulated after iron chelation and down-regulated by iron supplementation. These results are important for understanding the mechanisms of iron depletion-mediated cell cycle arrest, DNA damage repair, and apoptosis.

Cellular iron depletion stimulates the JNK and p38 MAPK signaling transduction pathways, dissociation of ASK1-thioredoxin, and activation of ASK1.

The role of signaling pathways in the regulation of cellular iron metabolism is becoming increasingly recognized. Iron chelation is used for the treatment of iron overload but also as a potential strategy for cancer therapy, because iron depletion results in cell cycle arrest and apoptosis. This study examined potential signaling pathways affected by iron depletion induced by desferrioxamine (DFO) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). Both chelators affected multiple molecules in the mitogen-activated protein kinase (MAPK) pathway, including a number of dual specificity phosphatases that directly de-phosphorylate MAPKs. Examination of the phosphorylation of major MAPKs revealed that DFO and Dp44mT markedly increased phosphorylation of stress-activated protein kinases, JNK and p38, without significantly affecting the extracellular signal-regulated kinase (ERK). Redox-inactive DFO-iron complexes did not affect phosphorylation of JNK or p38, whereas the redox-active Dp44mT-iron complex significantly increased the phosphorylation of these kinases similarly to Dp44mT alone. Iron or N-acetylcysteine supplementation reversed Dp44mT-induced up-regulation of phospho-JNK, but only iron was able to reverse the effect of DFO on JNK. Both iron chelators significantly reduced ASK1-thioredoxin complex formation, resulting in the increased phosphorylation of ASK1, which activates the JNK and p38 pathways. Thus, dissociation of ASK1 could serve as an important signal for the phosphorylation of JNK and p38 activation observed after iron chelation. Phosphorylation of JNK and p38 likely play an important role in mediating the cell cycle arrest and apoptosis induced by iron depletion.

The medicinal chemistry of novel iron chelators for the treatment of cancer.

Cancer is one of the leading causes of death worldwide and there is an increasing need for novel anti-tumor therapeutics with greater selectivity and potency. A new strategy in the treatment of cancer has focused on targeting an essential cell metabolite, iron (Fe). Iron is vital for cell growth and metabolism, forming a crucial component of the active site of ribonucleotide reductase (RR), the rate-limiting enzyme in DNA synthesis. Cancer cells in particular require large amounts of Fe to proliferate, making them more susceptible to the Fe deficiency caused by Fe chelators. Beginning with primordial siderophores, Fe chelators have since evolved to a new generation of potent and efficient anti-cancer agents. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands that demonstrate marked and selective anti-tumor activity both in vitro and in vivo against a wide spectrum of tumors. The mechanism of action of these novel ligands includes alterations in the expression of key regulatory molecules as well as the generation of redox active Fe complexes. Interestingly, non-synthetic Fe chelators including silybin and curcumin, both of which are derived from plants, also have vast potential in the treatment of cancer. This review explores the development of novel Fe chelators for the treatment of cancer and their mechanisms of action.

Synthetic and natural products as iron chelators.

An evaluation of existing and proposed Fe chelators, both synthetic and natural products, for the treatment of Fe-overload disease must address a number of issues. There are fundamental parameters that determine the efficacy of a drug: absorption, distribution, metabolism, clearance and toxicity. However, the administration of chelator for Fe overload aims to generate Fe complexes in vivo that are able to be excreted. Hence, the chemical and pharmacological properties of the complexes formed are equally important as the chelators themselves. The redox properties of the Fe complexes formed is particularly relevant to their toxicity. If both Fe(II) and Fe(III) oxidation states of the complexes are biologically accessible, then there is potential for the auto-catalytic production of deleterious free radicals, by Fenton-type chemistry. In addition, since the burden of Fe overload disease falls predominantly on some of the poorest economies, the cost of a drug must be considered, as well as the mode of delivery. There are also possible issues with the use of naturally occurring ligands, which may form Fe complexes capable of being utilised by opportunistic bacteria. This review will concentrate on recent developments in our chemical understanding of existing chelators approved or proposed for use and will also consider some of the candidates from natural sources that have been recently proposed.

The iron complex of Dp44mT is redox-active and induces hydroxyl radical formation: an EPR study.

Iron chelation therapy was initially designed to alleviate the toxic effects of excess iron evident in iron-overload diseases. However, some iron chelator-metal complexes have also gained interest due to their high redox activity and toxicological properties that have potential for cancer chemotherapy. This communication addresses the conflicting results published recently on the ability of the iron chelator, Dp44mT, to induce hydroxyl radical formation upon complexation with iron (B.B. Hasinoff and D. Patel, J Inorg. Biochem.103 (2009), 1093-1101). This previous study used EPR spin-trapping to show that Dp44mT-iron complexes were not able to generate hydroxyl radicals. Here, we demonstrate the opposite by using the same technique under very similar conditions to show the Dp44mT-iron complex is indeed redox-active and induces hydroxyl radical formation. This was studied directly in an iron(II)/H(2)O(2) reaction system or using a reducing iron(III)/ascorbate system implementing several different buffers at pH 7.4. The demonstration by EPR that the Dp44mT-iron complex is redox-active confirms our previous studies using cyclic voltammetry, ascorbate oxidation, benzoate hydroxylation and a plasmid DNA strand-break assay. We discuss the relevance of the redox activity to the biological effects of Dp44mT.

Future of toxicology--iron chelators and differing modes of action and toxicity: the changing face of iron chelation therapy.

Iron (Fe) chelation therapy was initially designed to alleviate the toxic effects of excess Fe evident in Fe-overload diseases. However, the novel toxicological properties of some Fe chelator-metal complexes have shifted appreciable focus to their application in cancer chemotherapy. Redox-inactive Fe chelator complexes are well suited for the treatment of Fe-overload diseases, whereas Fe chelator complexes with high redox activity have shown promising results as chemotherapeutics against cancer. Within this perspective, we discuss the different modes of action and toxicological profiles of Fe chelators, including analogues of 2-pyridylcarboxaldehyde isonicotinoyl hydrazone, di-2-pyridylketone isonicotinoyl hydrazone, di-2-pyridylketone thiosemicarbazone, and the clinically trialed chelator 3-aminopyridine-2-carboxaldehyde thiosemicarbazone. The potential application of these agents in the changing face of Fe chelation therapy is discussed.

A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics.

Novel chemotherapeutics with marked and selective antitumor activity are essential to develop, particularly those that can overcome resistance to established therapies. Iron (Fe) is critical for cell-cycle progression and DNA synthesis and potentially represents a novel molecular target for the design of new anticancer agents. The aim of this study was to evaluate the antitumor activity and Fe chelation efficacy of a new class of Fe chelators using human tumors. In this investigation, the ligands showed broad antitumor activity and could overcome resistance to established antitumor agents. The in vivo efficacy of the most effective chelator identified, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT), was assessed by using a panel of human xenografts in nude mice. After 7 weeks, net growth of a melanoma xenograft in Dp44mT-treated mice was only 8% of that in mice treated with vehicle. In addition, no differences in these latter animals were found in hematological indices between Dp44mT-treated mice and controls. No marked systemic Fe depletion was observed comparing Dp44mT- and vehicle-treated mice, probably because of the very low doses required to induce anticancer activity. Dp44mT caused up-regulation of the Fe-responsive tumor growth and metastasis suppressor Ndrg1 in the tumor but not in the liver, indicating a potential mechanism of selective anticancer activity. These results indicate that the novel Fe chelators have potent and broad antitumor activity and can overcome resistance to established chemotherapeutics because of their unique mechanism of action.

More roles for selenoprotein P: local selenium storage and recycling protein in the brain.

How is the essential micronutrient, selenium (Se), transported in the serum and then donated to tissues? In this issue of the Biochemical Journal, Schweizer and colleagues demonstrate, using conditional and total mouse knockout models, that SePP (selenoprotein P) is the major transporter of Se in the serum. Moreover, in the sanctuary area of the brain, SePP was shown to play a hitherto unexpected role as a local Se storage and recycling protein that directly maintains brain Se levels. Considering the function of Se in normal brain metabolism, these results are crucial for our understanding of the role of selenoproteins in redox regulation, antioxidant defences, thyroid hormone metabolism and the development of neurodegenerative conditions.

Interactions of the pyridine-2-carboxaldehyde isonicotinoyl hydrazone class of chelators with iron and DNA: implications for toxicity in the treatment of iron overload disease.

Iron chelation therapy for the management of iron-overload disease is dominated by desferrioxamine (DFO). However, treatment using DFO is very arduous. Recently, novel Fe chelators of the pyridine-2-carboxaldehyde isonicotinoyl hydrazone (PCIH) class have shown high chelation efficacy and the potential to replace DFO. A critical consideration in the design of alternatives to DFO is that the chelator forms a redox-inert Fe complex. In the present study, the participation of Fe complexes in redox reactions has been investigated. Ascorbate oxidation in the presence of Fe(III) or benzoate hydroxylation in the presence of Fe(II) was not enhanced by the PCIH analogues. However, redox-induced DNA strand breaks were observed with these ligands under highly oxidizing conditions in the presence of Fe(II) and hydrogen peroxide. Experiments then examined the interactions of the PCIH analogues with DNA, and this was found to be weak. Considering this, we suggest that under extreme conditions seen in the DNA-strand break assay, weak DNA-binding may potentiate the redox activity of the PCIH analogues. However, importantly, in contrast to naked plasmid DNA, DNA damage by these chelators using intact human cells was not significant. Collectively, our results support the potential of the PCIH analogues for the treatment of Fe overload.

Beta-thalassaemia: emergence of new and improved iron chelators for treatment.

Beta-thalassaemia is an inherited blood disorder which through repeated blood transfusions and enhanced iron uptake from the gastrointestinal tract, results in marked iron overload. Untreated, the iron accumulation results in the dysfunction of vital organs such as the heart and liver. At present, the most effective treatment for beta-thalassaemia is the use of the iron chelator, desferrioxamine, which is expensive, orally inactive and requires long subcutaneous infusions. In this concise review, we will focus on novel chelators which show therapeutic potential to replace desferrioxamine. Furthermore, we will discuss the potential of combined iron chelation therapy and the principle that, in the future, the use of more than just one chelator may be beneficial in tailoring individual iron chelation regimens.

Examination of the antiproliferative activity of iron chelators: multiple cellular targets and the different mechanism of action of triapine compared with desferrioxamine and the potent pyridoxal isonicotinoyl hydrazone analogue 311.

PURPOSE: Tumors are sensitive to iron (Fe) chelation therapy with the clinically used chelator desferrioxamine (DFO). Recently, the potent inhibitor of ribonucleotide reductase, Triapine, has entered clinical trials as an anticancer agent. This compound is a potential Fe chelator, but despite this, no investigations have examined its effect on cellular Fe metabolism. This is essential for understanding its mechanism of action and clinical effects. EXPERIMENTAL DESIGN: We compared the effect of Triapine with DFO, and also with the novel Fe chelator, 311, which shows marked antiproliferative activity. This latter ligand was relevant to compare, because it is tridentate like Triapine and shares structural similarity. We assessed the effects of chelators on proliferation, Fe uptake, Fe efflux, the expression of cell cycle control molecules, and iron-regulatory protein-RNA-binding activity. Redox activity was determined by ascorbate oxidation, benzoate hydroxylation, plasmid DNA degradation, and the precipitation of cellular DNA. These studies have been performed using several neuroepithelioma and neuroblastoma cell lines and a variety of normal cell types including fibroblasts, umbilical vein endothelial cells, skeletal muscle cells, monocyte-derived macrophages, and bone marrow stem cells. RESULTS: Triapine was twice as effective as DFO at mobilizing (59)Fe from prelabeled cells but was much less efficient than 311. In terms of preventing (59)Fe uptake from Tf, Triapine and DFO had similar activity, having far less efficacy than 311. All three of the chelators showed greater activity against the proliferation of neoplastic than of normal cells, the effect of 311 and Triapine being similar and these two chelators being significantly (P < 0.0001) more active than DFO. Complexation of Triapine with Fe had no appreciable effect on its antiproliferative activity, whereas addition of Fe totally inhibited the effects of DFO and 311. Furthermore, the Triapine Fe complex was shown to be redox active. CONCLUSION: The cytotoxic mechanism of action of Triapine was different from that of DFO and 311, with the combined action of Fe chelation and free radical generation being involved.