Recent advances in cancer treatment by iron chelators.

The development of new therapeutic alternatives for cancers is a major public health priority. Among the more promising approaches, the iron depletion strategy based on metal chelation in the tumoral environment has been particularly studied in recent decades. After a short description of the importance of iron for cancer cell proliferation, we will review the different iron chelators developed as potential chemotherapeutics. Finally, the recent efforts to vectorize the chelating agents specifically in the microtumoral environment will be discussed in detail.

Synthesis and biological properties of Quilamines II, new iron chelators with antiproliferative activities.

To selectively target tumor cells expressing an overactive Polyamine Transport System (PTS), we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, derived from the lead compound HQ1-44, which we named Quilamines II. The structures of four new antiproliferative agents were developed. They differ in the size of the linker (HQ0-44 and HQ2-44) or in the nature of the linker (HQCO-44 and HQCS-44) between a hydroxyquinoline moiety (HQ) and a homospermidine (44) chain, the best polyamine vector. The Quilamines II were obtained after 6 to 9 steps by Michael addition, peptide linkage, and reductive amination or by using the Willgerodt-Kindler reaction. The biological evaluation of these second-generation Quilamines showed that modifying the size of the linker increased the selectivity of these compounds for the PTS. In addition, measurement of the toxicity of Quilamines HQ0-44 and HQ2-44 highlighted their marked antiproliferative nature on several cancerous cell lines as well as a differential activity on nontransformed cells (fibroblasts). In contrast, Quilamines HQCO-44 and HQCS-44 presented low selectivity for the PTS, probably due to a loss of electrostatic interaction. We also demonstrated that the HCT116 cell line, originating from a human colon adenocarcinoma, was the most responsive to the various Quilamines. As deduced from the calcein and HVA assays, the higher iron chelating capacity of HQ1-44 could explain its higher antiproliferative efficiency.

Involvement of polyamines in iron(III) transport in human intestinal Caco-2 cell lines.

Natural polyamines such as putrescine (Put), spermidine (Spd), and spermine (Spm), which are present in the human diet in large amounts, associated with their active transporter, are assumed to play a role in non-heme iron uptake and iron bioavailability from nutrients. Enterocytes and hepatocytes play pivotal roles in the regulation of body iron homeostasis. In this study, we report the effects of natural polyamines on iron transport in the Caco-2 cell line. In enterocyte-like Caco-2 cells, polyamines did not significantly modulate the transepithelial iron flux across the cell monolayer cultured on permeable membranes. In contrast, Spd, Spm, and to a lesser extent, Put were shown to activate Caco-2 cell iron uptake and to induce an increase in the ferritin level. This iron co-transport in enterocytes, which involved an interaction between iron and polyamine then cell uptake of the polyamine-iron complexes by the polyamine transport system, was more pronounced in proliferating than in differentiated Caco-2 cells. Moreover, it was observed at physiological concentrations of both polyamines and iron. It could thus play a role in the rapid renewal of enterocytes. These data suggest the involvement of polyamines as components of the pool of transferrin-independent iron-chelating vectors. Further investigations are needed to demonstrate their biological relevance in physiological situations.

Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation.

Iron chelation in tumoral cells has been reported as potentially useful during antitumoral treatment. Our aim was to develop new polyaminoquinoline iron chelators targeting tumoral cells. For this purpose, we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, which we named Quilamines, based on an 8-hydroxyquinoline (8-HQ) scaffold linked to linear polyamine vectors. These were designed to target tumor cells expressing an overactive polyamine transport system (PTS). A set of Quilamines bearing variable polyamine chains was designed and assessed for their ability to interact with iron. Quilamines were also screened for their cytostatic/cytotoxic effects and their selective uptake by the PTS in the CHO cell line. Our results show that both the 8-HQ moiety and the polyamine part participate in the iron coordination. HQ1-44, the most promising Quilamine identified, presents a homospermidine moiety and was shown to be highly taken up by the PTS and to display an efficient antiproliferative activity that occurred in the micromolar range. In addition, cytotoxicity was only observed at concentrations higher than 100 µM. We also demonstrated the high complexation capacity of HQ1-44 with iron while much weaker complexes were formed with other cations, indicative of a high selectivity. We applied the density functional theory to study the binding energy and the electronic structure of prototypical iron(III)-Quilamine complexes. On the basis of these calculations, Quilamine HQ1-44 is a strong tridentate ligand for iron(III) especially in the form of a 1:2 complex.

Ethanol effect on cell proliferation in the human hepatoma HepaRG cell line: relationship with iron metabolism.

BACKGROUND: Alcoholism increases the risk of cirrhosis and/or hepatocellular carcinoma development. Iron, like ethanol, modulates the cell growth. However, the relationship between alcohol and iron toward hepatocyte proliferation has not been clearly elucidated. The purpose of this study was to evaluate, in the human HepaRG cell line model, the impact of ethanol on hepatocyte proliferation in relation to modulations of iron metabolism and the protective effect of iron metabolism manipulation by chelators in alcohol liver diseases. METHODS: The human hepatoma HepaRG cell line model was used. Cell viability was determined by measuring succinate dehydrogenase activity, total protein level by the Bradford method. DNA synthesis was evaluated by [(3)H]-methyl thymidine incorporation. Cytotoxicity was studied by release of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) in culture medium and apoptosis by measuring caspase 3/7 activity. Gene expression was analyzed by RT-qPCR. Total iron, soluble transferrin receptor, and ferritin levels were, respectively, measured by colorimetrical, immuno-nephelometrical, and immuno-turbidimetrical methods. Intracellular iron uptake and accumulation was examined by radionuclide (55)Fe (III) measurement and Perls staining. RESULTS: Results showed that ethanol decreased all the parameters associated with HepaRG cell proliferation (cell viability, total protein levels, and DNA synthesis) in a dose- and time-dependent manner. This effect was accompanied by cytotoxicity and apoptosis as evaluated by a significant increase in extracellular enzymes (LDH, AST, ALT) and caspase 3/7 activity, respectively. Ethanol exposure was accompanied by an increased cellular iron uptake, together with increased expression of genes involved in iron transport and storage such as l-ferritin, Divalent Metal transporter 1, transferrin, transferrin receptor 1, and ceruloplasmin. Ethanol impact was intensified by iron-citrate and decreased by iron chelators when added to the culture medium. CONCLUSIONS: The results indicated that (i) ethanol-induced iron metabolism dysfunction could be one of the underlying mechanisms of ethanol antiproliferative effect and (ii) exogenous iron may accentuate ethanol hepatoxicity. These data suggest that iron metabolism manipulation by chelators may be a useful therapeutic approach in alcohol-related liver diseases.

Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes. Part II.

Cell cycle progression is dependent on the intracellular iron level and chelators can lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of some new synthetic calix[4]arene podands bearing diamino-tetraesters, diamino-tetraalcohols, diamino-tetraacid and tetraaryloxypentoxy groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670A (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in the human hepatocarcinoma HepaRG cell cultures using cell nuclei counting after staining with the DNA intercalating fluorescence dye, Hoechst 33342. Their cytotoxicity was evaluated by the extracellular LDH activity. Preliminary results indicated that their antiproliferative effect was mainly due to their cytotoxicity. The efficiency of these compounds, being comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that the new substituted calix[4]arenes could open the way to valuable new approaches for medicinal chemistry scaffolding.

Synthesis and biological properties of iron chelators based on a bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamide or -thiocarboxamide (BHPTC) scaffold.

Bis-2-(2-hydroxy-phenyl)-thiazole-4-carboxamides and -thiocarboxamides (BHPTCs) form a family of gemini hexacoordinated bis-tridentate chelating scaffolds. Four molecules were synthesized and shown to chelate iron(III) efficiently with a 1:1 stoichiometry. A dithioamide BHPTC displayed promising antiproliferative activity in several cancerous cell lines, making this molecule an interesting lead compound for the design of new iron-chelating anticancer drugs. Conversely, diamide BHPTCs had significant cytoprotective activity against iron overload in HepaRG cells in vitro, and were as efficient as and less toxic than deferoxamine B (DFO).

Effects of deferasirox and deferiprone on cellular iron load in the human hepatoma cell line HepaRG.

Two oral chelators, CP20 (deferiprone) and ICL670 (deferasirox), have been synthesized for the purpose of treating iron overload diseases, especially thalassemias. Given their antiproliferative effects resulting from the essential role played by iron in cell processes, such compounds might also be useful as anticancer agents. In the present study, we tested the impact of these two iron chelators on iron metabolism, in the HepaRG cell line which allowed us to study proliferating and differentiated hepatocytes. ICL670 uptake was greater than the CP20 uptake. The iron depletion induced by ICL670 in differentiated cells increased soluble transferrin receptor expression, decreased intracellular ferritin expression, inhibited (55)Fe (III) uptake, and reduced the hepatocyte concentration of the labile iron pool. In contrast, CP20 induced an unexpected slight increase in intracellular ferritin, which was amplified by iron-treated chelator exposure. CP20 also promoted Fe(III) uptake in differentiated HepaRG cells, thus leading to an increase of both the labile pool and storage forms of iron evaluated by calcein fluorescence and Perls staining, respectively. In acellular conditions, compared to CP20, iron removing ability from the calcein-Fe(III) complex was 40 times higher for ICL670. On the whole, biological responses of HepaRG cells to ICL670 treatment were characteristic of expected iron depletion. In contrast, the effects of CP20 suggest the potential involvement of this compound in the iron uptake from the external medium into the hepatocytes from the HepaRG cell line, therefore acting like a siderophore in this cell model.

Antiproliferative effect on HepaRG cell cultures of new calix[4]arenes.

Cell cycle progression is dependent on the intracellular iron level, and chelators lead to iron depletion and decrease cell proliferation. This antiproliferative effect can be inhibited by exogenous iron. In this work, we present the synthesis of new synthetic calix[4]arene podands bearing alkyl acid and alkyl ester groups at the lower rim, designed as potential iron chelators. We report their effect on cell proliferation, in comparison with the new oral chelator ICL670 (4-[3,5-bis-(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid). The antiproliferative effect of these new compounds was studied in human hepatocarcinoma HepaRG cell cultures using the MTT assay. Their cytotoxicity was evaluated by extracellular LDH activity. Preliminary results indicate that their antiproliferative effect is due to their cytotoxicity. The efficiency of these compounds, comparable to that of ICL670, was independent of iron depletion. This effect remains to be further explored. Moreover, it also shows that novel substituted calix[4]arenes could open the way to new valuable medicinal chemistry scaffolding.

Hemochromatosis: a model of metal-related human toxicosis.

Many environmental agents, such as excessive alcohol intake, xenobiotics, and virus, are able to damage the human body, targeting especially the liver. Metal excess may also assault the liver. Thus, chronic iron overload may cause, especially when associated with cofactors, diffuse organ damage that is a source of significant morbidity and mortality. Iron excess can be either of acquired (mostly transfusional) or of genetic origin. Hemochromatosis is the archetype of genetic iron overload diseases and represents a serious health problem. A better understanding of iron metabolism has deeply modified the hemochromatosis field which today benefits from much more efficient diagnostic and therapeutic approaches.

Protective effect of monosialoganglioside GM1 against chemically induced apoptosis through targeting of mitochondrial function and iron transport.

Exogenous treatment with monosialoganglioside GM1 has been described to afford protection against different apoptotic insults. However, the underlying mechanisms remain to be determined. In this study, we focused on the effect of GM1 on the apoptotic cascade induced by benzo[a]pyrene (B[a]P) in rat hepatic F258 epithelial cells. We first demonstrated that a co-treatment with GM1 (80 microM) reduced B[a]P (50 nM)-induced apoptosis as evidenced by a decrease of both cell population exhibiting nuclear fragmentation and caspase 3 cleavage and activity. We next showed that the p53 phosphorylation and nuclear translocation as well as the intracellular alkalinization related to Na+/H+ exchanger 1 (NHE1) activation, two early events of the apoptosis induced by B[a]P, were not inhibited by GM1. In contrast, the late mitochondria-dependent acidification elicited by B[a]P was inhibited by GM1 co-treatment, and an inhibition of the oxidative stress was also observed. Because GM1 has been shown to reduce the low-molecular weight iron content related to ethanol-induced oxidative stress, we finally investigated the involvement of iron under our conditions. Using the two iron chelators deferiprone and desferrioxamine, we clearly showed that iron played an important role in B[a]P-induced apoptosis in F258 cells, and that B[a]P-treatment resulted in a significant GM1-sensitive increase in (55)Fe uptake. In conclusion, our results indicate that exogenous GM1 partly prevents B[a]P-induced apoptosis by interfering with mitochondria-related intracellular acidification and iron transport.

Membrane fluidity changes are associated with benzo[a]pyrene-induced apoptosis in F258 cells: protection by exogenous cholesterol.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]yrene (B[a]P) constitute a widely distributed class of environmental pollutants, responsible for highly toxic effects. Elucidating the intracellular mechanisms of this cytotoxicity thus remains a major challenge. Besides the activation of the p53 apoptotic pathway, we have previously found in F258 hepatic cells that the B[a]P (50 nM)-induced apoptosis was also dependent upon the transmembrane transporter NHE1, whose activation might result from membrane alterations in our model. We here demonstrate that: (1) B[a]P induces a membrane fluidization surprisingly linked to NHE1 activation; (2) membrane stabilization by exogenous cholesterol protects cells from B[a]P-induced apoptosis, via an effect on late acidification and iron uptake.

The new orally active iron chelator ICL670A exhibits a higher antiproliferative effect in human hepatocyte cultures than O-trensox.

By comparing the antiproliferative effect of the iron chelators ICL670A and O-trensox in the human hepatoma cell line HUH7 and human hepatocyte cultures, we have shown that ICL670A decreased cell viability, inhibited DNA replication and induced DNA fragmentation more efficiently than O-trensox. O-trensox and ICL670A induced a cell cycle blockade in G0-G1 and S phases respectively. In parallel, ICL670A inhibited polyamine biosynthesis by decreasing ornithine decarboxylase and spermidine/spermine N(1)-acetyltransferase activities. O-trensox increased polyamine biosynthesis and particularly putrescine level by stimulating spermidine-spermine N(1)-acetyltransferase activity which could activate the polyamine retro-conversion pathway. Moreover, the two chelators exhibit some cytotoxic effect in the two culture models; ICL670A was more cytotoxic than O-trensox and higher concentrations of the two chelators were necessary to induce a cytotoxicity in primary cultures versus hepatoma cells. These results suggested that ICL670A has the most efficient antitumoral effect, blocks cell proliferation by a pathway different of O-trensox and may constitute a potential drug for anticancer therapy.

[Normal iron metabolism]. / Métabolisme du fer chez le sujet normal.

Normal iron metabolism is highly regulated and takes a crucial role in the maintenance of cell functions. The plasmatic iron bioavailability control is a key step of this metabolism which involves numerous proteins implicated at various levels, including the digestive iron absorption by enterocytes, and iron release from macrophages. These two phenomenons are modulated in a coordonated fashion by the plasmatic level of hepcidin, a peptide mainly synthetized by the liver, secreted in plasma and modulating the expression of ferroportin, the cellular exporter of iron, and thus the iron egress. Numerous factors are able to modulate the hepcidin expression, including iron status, erythropoietic activity, inflammation and hepatic status which are already identified. Abnormalities occurring in the regulation of hepcidin expression may favour the development of iron metabolism disturbance, including systemic iron overload or relative iron deficiency. The use of hepcidin for diagnostic purpose or as a therapeutic target remains to be determined.

[Genetic iron overload diseases: a deeply changing world]. / Les surcharges génétiques en fer: un monde en pleine mutation.

Iron overload diseases are a quickly and deeply changing world, due to major advances in genetics and molecular biology. Five main entities are concerned: a frequent one, namely HFE-related or type1 haemochromatosis, and four rare or exceptional diseases which are types 2, 3 and 4 haemochromatosis and aceruloplasminemia. Increased duodenal iron absorption and enhanced macrophagic iron recycling, both due to hypo-hepcidinemia, account for the development of cellular excess in types 1, 2, 3 haemochromatosis whereas decreased cellular iron egress is the main explanation for type 4 haemochromatosis and aceruloplasminemia. Non-transferrin bound iron plays an important role in cellular iron excess and damage. Phlebotomies remain an essential therapeutic tool but the improved understanding of the intimate mechanisms underlying these diseases open the road for innovative therapeutic approaches.

Regulation of arginase II by interferon regulatory factor 3 and the involvement of polyamines in the antiviral response.

The innate antiviral response requires the induction of genes and proteins with activities that limit virus replication. Among these, the well-characterized interferon beta (IFNB) gene is regulated through the cooperation of AP-1, NF-kappaB and interferon regulatory factor 3 (IRF-3) transcription factors. Using a constitutively active form of IRF-3, IRF-3 5D, we showed previously that IRF-3 also regulates an IFN-independent antiviral response through the direct induction of IFN-stimulated genes. In this study, we report that the arginase II gene (ArgII) as well as ArgII protein concentrations and enzymatic activity are induced in IRF-3 5D-expressing and Sendai virus-infected Jurkat cells in an IFN-independent manner. ArgII is a critical enzyme in the polyamine-biosynthetic pathway. Of the natural polyamines, spermine possesses antiviral activity and mediates apoptosis at physiological concentrations. Measurement of intracellular polyamine content revealed that expression of IRF-3 5D induces polyamine production, but that Sendai virus and vesicular stomatitis virus infections do not. These results show for the first time that the ArgII gene is an early IRF-3-regulated gene, which participates in the IFN-independent antiviral response through polyamine production and induction of apoptosis.

Quilamine HQ1-44, an iron chelator vectorized toward tumor cells by the polyamine transport system, inhibits HCT116 tumor growth without adverse effect.

Tumor cell growth requires large iron quantities and the deprivation of this metal induced by synthetic metal chelators is therefore an attractive method for limiting the cancer cell proliferation. The antiproliferative effect of the Quilamine HQ1-44, a new iron chelator vectorized toward tumor cells by a polyamine chain, is related to its high selectivity for the Polyamine Transport System (PTS), allowing its preferential uptake by tumoral cells. The difference in PTS activation between healthy cells and tumor cells enables tumor cells to be targeted, whereas the strong dependence of these cells on iron ensures a secondary targeting. Here, we demonstrated in vitro that HQ1-44 inhibits DNA synthesis and cell proliferation of HCT116 cells by modulating the intracellular metabolism of both iron and polyamines. Moreover, in vivo, in xenografted athymic nude mice, we found that HQ1-44 was as effective as cis-platin in reducing HCT116 tumor growth, without its side effects. Furthermore, as suggested by in vitro data, the depletion in exogenous or endogenous polyamines, known to activate the PTS, dramatically enhanced the antitumor efficiency of HQ1-44. These data support the need for further studies to assess the value of HQ1-44 as an adjuvant treatment in cancer.

Effect of Caseinophosphopeptides from αs- and ß-Casein on Iron Bioavailability in HuH7 Cells.

Two pools of caseinophosphopeptides (CPPs) obtained from αs- and ß-casein fractions (α-CPPs and ß-CPPs) were characterized. A total of 16 CPPs were identified in the α-CPPs pool, 9 of them derived from αs1-casein and 7 from αs2-casein. A total of 18 CPPs were identified in the ß-CPPs pool. Four of the identified CPPs contained the characteristic phosphoseryl-glutamic acid cluster SpSpSpEE. Calcein assay was used to compare the iron-binding capacity of the α- and ß-CPPs pools. At the concentration of 12.5 µM CPPs used in the iron bioavailability assays, ß-CPPs pools show greater iron-binding capacity than α-CPPs pools. HuH7 human hepatoma cells show many differentiated functions of liver cells in vivo and can be used to evaluate iron bioavailability (ferritin content and soluble transferrin receptor) from Fe-α-CPPs and Fe-ß-CPPs complexes. The α-CPPs and ß-CPPs pools did not improve ferritin content or soluble transferrin receptor in HuH7 cells.

Polyamine modulation of iron uptake in CHO cells.

Polyamines are ubiquitous molecules, which, like iron, are essential for cell growth. All eukaryotic cells are equipped with a specific polyamine transport system (PTS). Polyamines have primary and secondary amino groups which chelate bivalent metal cations such as Fe and Cu. In the present study, we investigated the potential contribution of naturally occurring polyamines and their active transport system to iron uptake. In presence of subtoxic Fe(III) (10microM), treatment of CHO cells with spermine, and to a lesser extent with spermidine (10-100microM), resulted in a marked cytotoxic effect. This cytotoxicity was prevented by the addition of an iron-chelator, deferioxamine, and was not observed in CHO-MG cells, a mutant cell line devoid of polyamine transport activity. Experiments using 14C-polyamines and 55Fe(III) revealed that these toxic effects were related to polyamine-modulation of iron uptake, and were dependent on the presence of the active PTS. These results demonstrated active uptake of polyamine-iron complexes via the PTS. The number of amino groups affected the efficacy of the studied natural polyamines to transport iron via the PTS. Spermine, a tetramine, was more efficient than the triamine spermidine. Co-transport of iron by the diamine putrescine was not observed. These results demonstrate that the cell polyamine transport system is a potential cell entry pathway for iron. The studied polyamines, spermine and spermidine, may be components of the pool of transferrin-independent iron-chelating vectors, which have recently attracted the attention of many investigators.

Iron mobilization, cytoprotection, and inhibition of cell proliferation in normal and transformed rat hepatocyte cultures by the hydroxypyridinone CP411, compared to CP20: a biological and physicochemical study.

The present study analyzes the iron mobilization, the cytoprotective, and the antiproliferative effects of the lipophilic hydroxypyridinone CP411, in comparison with the hydrophilic chelator CP20 or deferiprone used in the treatment of iron overload. Primary rat hepatocyte cultures and the rat hepatoma cell line Fao were used. Chelator cell uptake was evaluated by mass spectrometry in the two models. This method was also used to investigate the stability of the chelators in an acellular system as well as their scavenging and chelating effects against the hydroxyl radical generated by the Fenton reaction. The iron mobilization and the cytoprotective effects of the chelators were evaluated in primary cultures by measuring respectively 55Fe and lactate dehydrogenase release in the culture medium. The antiproliferative effect of the chelators was studied using the Fao cell line and measuring DNA synthesis by thymidine incorporation and DNA content by flow cytometry. We observed that CP411 entered the hepatocytes and the Fao cells respectively 4 and 13 times more than CP20. CP411 was 2.5 times more effective than CP20 to mobilize iron from preloaded hepatocytes. Pretreatment of the hepatocytes with CP20 or CP411 decreased the toxic effect of iron and CP411 was 1.6 times more effective than CP20. A dose-dependent decrease of DNA synthesis, correlated to an accumulation of cells in S phase, was observed in the Fao cell line in the presence of CP411, while CP20 was without effect. CP411 effect was inhibited by addition of iron simultaneously with the chelator, the addition of Zn or Cu was without effect. The inhibitory effect of CP411 was reversible since, 24hr after removal of the chelator, DNA replication reached the control level. The results show that CP411 is more efficient to protect the hepatocyte from the toxic effect of iron load and to inhibit tumor cell proliferation. Its higher efficiency may result from its better cell uptake since equimolar solutions of the two chelators in an acellular system exhibit the same ability to inhibit the Fenton reaction.