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 ⁵⁵Fe (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.     

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).     

Modulation of cell proliferation and polyamine metabolism in rat liver cell cultures by the iron chelator O-trensox

The antiproliferative effects of the iron chelator O-trensox and the ornithine-decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) were characterized in the rat hepatoma cell line FAO, the rat liver epithelial cell line (RLEC) and the primary rat hepatocyte cultures stimulated by EGF. We observed that O-trensox and DFMO decreased cell viabilty and DNA replication in the three culture models. The cytostatic effect of O-trensox was correlated to a cytotoxicity, higher than for DFMO, and to a cell cycle arrest in G0/G1 or S phases. Moreover, O-trensox and DFMO decreased the intracellular concentration of spermidine in the three models without changing significantly the spermine level. We concluded that iron, but also polyamine depletion, decrease cell growth. However, the drop in cell proliferation obtained with O-trensox was stronger compared to DFMO effect. Altogether, our data provide insights that, in the three rat liver cell culture models, the cytostatic effect of the iron chelator O-trensox may be the addition of two mechanisms: iron and polyamine depletion.     

The intracellular iron sensor calcein is catalytically oxidatively degraded by iron(II) in a hydrogen peroxide-dependent reaction

The fluorescent metal chelating dye calcein is used to obtain an estimate of cellular iron levels and to measure the kinetics of the entry of chelators and chelating drugs into cells. Under reducing conditions in the presence of ascorbic acid, such as that would be present in the cell, the Fe(II)-calcein complex was rapidly formed with a rate constant of 3 x 10(5) M(-1) s(-1). A slower iron-dependent catalytic degradation of calcein also occurred that resulted in the formation of a non-fluorescent calcein product. The Fe(II)-catalyzed degradation of calcein was largely, but not completely, prevented by catalase. Electron paramagnetic resonance spin trapping experiments showed that the Fe(II)-calcein complex promoted formation of hydroxyl or a hydroxyl radical-like species. Together these results indicated that Fe(II) catalyzed the degradation of calcein through both hydrogen peroxide, and to a lesser extent, non-hydrogen peroxide-dependent pathways. The iron-calcein complexes that were responsible for the degradation of calcein were likely high valence oxidizing iron-oxo species such as perferryl or ferryl complexes that were redox cycled by ascorbic acid. Thus, the use of calcein as an intracellular iron-sensing indicator may yield misleading results due to its degradation under certain conditions.     

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.     

Antiproliferative activity of novel derivative of thiopyran on breast and colon cancer lines and DNA binding

Dimethyl-5-acetyl-4-methyl-6-(4-methylphenylimino)-6H-thiopyran-2,3-dicarboxylate (4) has been synthesized and its antiproliferative activity is reported here. Compound 4 inhibited the growth of human colon cancer cell line HCT-15 with an IC(50) value of 3.5 μM and of breast cancer cell line MCF-7 with an IC(50) value of 1.5 μM in a dose/time-dependent manner using a sulforhodamine B assay. Moreover, suppression of clonogenic activity occurred after exposure to 4 at a concentration of 4 μM for HCT-15 and 1.7 μM for MCF-7. The results also showed tumor cell invasion through matrigel and cell adhesion. The effect of ligand complexation on DNA structure led to overall affinity constant of K(4-DNA)=9.8×10(4) M(-1).     

In vitro interaction between ceruloplasmin and human serum transferrin

The thermodynamics of the interactions of serum apotransferrin (T) and holotransferrin (TFe(2)) with ceruloplasmin (Cp), as well as those of human lactoferrin (Lf), were assessed by fluorescence emission spectroscopy. Cp interacts with two Lf molecules. The first interaction depends on pH and μ, whereas the second does not. Dissociation constants were as follows: K(11Lf) = 1.5 ± 0.2 μM, and K(12Lf) = 11 ± 2 μM. Two slightly different interactions of T or TFe(2) with Cp are detected for the first time. They are both independent of pH and μ and occur with 1:1 stoichiometry: K(1T) = 19 ± 7 μM, and K(1TFe2) = 12 ± 4 μM. These results can improve our understanding of the probable process of the transfer of iron from Cp to T in iron and copper transport and homeostasis.     

GalNAc/Gal-binding Rhizoctonia solani agglutinin has antiproliferative activity in Drosophila melanogaster S2 cells via MAPK and JAK/STAT signaling

Rhizoctonia solani agglutinin, further referred to as RSA, is a lectin isolated from the plant pathogenic fungus Rhizoctonia solani. Previously, we reported a high entomotoxic activity of RSA towards the cotton leafworm Spodoptera littoralis. To better understand the mechanism of action of RSA, Drosophila melanogaster Schneider S2 cells were treated with different concentrations of the lectin and FITC-labeled RSA binding was examined using confocal fluorescence microscopy. RSA has antiproliferative activity with a median effect concentration (EC(50)) of 0.35 μM. In addition, the lectin was typically bound to the cell surface but not internalized. In contrast, the N-acetylglucosamine-binding lectin WGA and the galactose-binding lectin PNA, which were both also inhibitory for S2 cell proliferation, were internalized whereas the mannose-binding lectin GNA did not show any activity on these cells, although it was internalized. Extracted DNA and nuclei from S2 cells treated with RSA were not different from untreated cells, confirming inhibition of proliferation without apoptosis. Pre-incubation of RSA with N-acetylgalactosamine clearly inhibited the antiproliferative activity by RSA in S2 cells, demonstrating the importance of carbohydrate binding. Similarly, the use of MEK and JAK inhibitors reduced the activity of RSA. Finally, RSA affinity chromatography of membrane proteins from S2 cells allowed the identification of several cell surface receptors involved in both signaling transduction pathways.     

Antiproliferative effects of some novel synthetic solanidine analogs on HL-60 human leukemia cells in vitro

There is increasing evidence of the direct antiproliferative effects of various steroidal structures, including cardenolides, steroidal alkaloids and sexual hormones. The aim of the present study was to characterize the antiproliferative effects of three synthetic solanidine analogs (1-3) on HL-60 human leukemia cells. The three compounds exerted similar cytostatic effects (IC(50) values: 1.27-2.94 μM after a 72-h exposure) and the most effective (2) was selected for further investigations. Incubation with compound 2 resulted in a marked chromatin condensation followed by a gradual increase in cell membrane permeability detected by Hoechst dye 33258-propidium iodide double staining. A flow cytometric analysis revealed a marked decrease in the G1 phase and substantial increases in the S and G2/M phases after 24-h incubation, while after 48 h the proportion of cells in the subG1 phase was increased significantly with a concomitant decrease in cells in the G1 and G2/M phases. Compound 2 at 6.0 μM significantly decreased the activity of ribonucleotide reductase and proved to be a potent antioxidant in the lipid peroxidation and DPPH assays (IC(50) values: 2.0 and 13.1 μM, respectively). The antiproliferative effect of the test compound on the non-cancerous human lung fibroblast cell line (MRC-5) was significantly weaker than that on the leukemia cells. These results lead to the conclusion that compound 2 induces a marked disturbance in the cell cycle, which is, at least partially, a consequence of the inhibition of DNA synthesis.     

Synthesis,Antiproliferative Evaluation,and Molecular Docking Study of Novel Longifolene-Derived Tetraline Fused Thiazole-Amide Derivatives

Twenty novel longifolene-derived tetraline fused thiazole-amide compounds were synthesized from longifolene, a renewable natural resource. Their structures were characterized by FT-IR, NMR, ESI-MS, and elemental analysis. The in vitro antiproliferative activity of these compounds against SK-OV-3 ovarian cancer cell lines, MCF-7 human breast cancer cell lines, HepG2 human liver cancer cell lines, A549 human lung adenocarcinoma cell lines, and T-24 human bladder cancer cell lines was tested by MTT assay. Compounds 6a – 6c displayed significant and broad-spectrum antiproliferative activity against almost the tested cancer cell lines with IC₅₀ in the range of 7.84 to 55.88 μM, of which compound 6c exhibited excellent antiproliferative activities with 7.84 μM IC₅₀ against SKOV-3, 13.68 μM IC₅₀ against HepG2, 15.69 μM IC₅₀ against A549, 19.13 μM IC₅₀ against MCF-7, and 22.05 μM IC₅₀ against T-24, showing better and broad-spectrum antiproliferative effect than that of the positive control 5-FU. Furthermore, the action model was analyzed by the molecular docking study. Some intriguing structure-activity relationships were found and discussed herein by DFT theoretical calculation.     

Click chemistry based multicomponent approach in the synthesis of spirochromenocarbazole tethered 1,2,3-triazoles as potential anticancer agents

A series of spirochromenocarbazole tethered 1,2,3-triazoles were synthesized via click chemistry based one-pot, five component reaction between N-propargyl isatins, malononitrile, 4-hydroxycarbazole, aralkyl halides and sodium azide using cellulose supported CuI nanoparticles (Cell-CuI NPs) as the heterogeneous catalyst. Antiproliferative activity of all the synthesized compounds was investigated against panel of cancer cell lines such as MCF-7, MDA-MB-231, HeLa, PANC-1, A-549, and THP-1. Many of the synthesized compounds exhibited good anti-proliferative activity against breast (MCF-7 and MDA-MB-231) and cervical (HeLa) cancer cells with IC₅₀ values less than 10 μM. In case of MCF-7 cells, among the nine compounds that showed good anti-proliferative activity, compounds 6f and 6j were found to be highly potent (IC₅₀ = 2.13 μM and 4.80 μM, respectively). In case of MDA-MB-231, three compounds (6k, 6j and 6s) showed antiproliferative activity amongst which 6k was the most potent one (IC₅₀ = 3.78 μM). On the other hand, in cervical cancer HeLa cells, compounds 6b, 6g, 6s and 6u showed excellent antiproliferative activity (IC₅₀ = 4.05, 3.54, 3.83, 3.35 μM, respectively). All the compounds were found to be nontoxic to the human umbilical vein endothelial cells (HUVECs). AO and EtBr staining and fluorescence microscopy studies of the active compounds (IC₅₀ < 5 μM) suggested that these compounds induce cell death by apoptosis.     

Synthesis,Antiproliferative and Cytotoxic Activities,DNA Binding Features and Molecular Docking Study of Novel Enamine Derivatives

Novel enamine derivatives were synthesized from the reaction of lactone and chalcones and their antiproliferative and cytotoxic activities against six cancer cell lines (e. g., HeLa, HT29, A549, MCF7, PC3 and Hep3B) and one normal cell lines (e. g., FL) were investigated along with their mode of interactions with CT‐DNA. Most of the enamine derivatives with IC₅₀ values of 86–168 μM demonstrated much stronger antiproliferative activity than the starting molecules against the cancer cells. While, among the enamine derivatives, four compounds displayed higher cytotoxic potency than the control drugs (5‐fluorouracil and cisplatin) against the Hep3B cell lines, these compounds did not exhibit any significant toxicity against normal cells, FL. The UV/VIS spectral data suggest that eight compounds cause hypochromism with a slight bathochromic shift (～6 nm), indicating that they bind to the DNA by way of an intercalative or minor groove binding mode. The binding constants of the compounds are in the range of 0.1×103 M^?1–2.3×104 M^?1. The antiproliferative activity of studied enamine derivatives could possibly be due to their DNA binding as well as their cytotoxic properties. In addition to these assays, the chalcones and enamine derivatives were investigated by molecular docking to calculate the synergistic effect of antiproliferative activities against six human cancer cell lines.     

Estradiol attenuation of β-amyloid-induced toxicity: A comparison of MTT and calcein AM assays

17β-estradiol (βE2) has been shown to attenuate the toxicity of β-amyloid peptides (Aβ) in neuronal cultures with the effective concentration of βE2 ranging from low nM to high μM. This study compares the effective neuroprotective concentration of βE2 against both Aβ-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective βE2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum βE2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM βE2 conferring significant protection in the calcein AM assay but 1 μM βE2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic Aβ concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM βE2 against H_{2 2} toxicity. These results indicate that low concentrations of βE2 can attenuate Aβ and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of βE2-mediated attenuation of Aβ toxicity.     

Iron specificity of a biosensor based on fluorescent pyoverdin immobilized in sol-gel glass

Two current technologies used in biosensor development are very promising: 1. The sol-gel process of making microporous glass at room temperature, and 2. Using a fluorescent compound that undergoes fluorescence quenching in response to a specific analyte. These technologies have been combined to produce an iron biosensor. To optimize the iron (II or III) specificity of an iron biosensor, pyoverdin (a fluorescent siderophore produced by Pseudomonas spp.) was immobilized in 3 formulations of porous sol-gel glass. The formulations, A, B, and C, varied in the amount of water added, resulting in respective R values (molar ratio of water:silicon) of 5.6, 8.2, and 10.8. Pyoverdin-doped sol-gel pellets were placed in a flow cell in a fluorometer and the fluorescence quenching was measured as pellets were exposed to 0.28 - 0.56 mM iron (II or III). After 10 minutes of exposure to iron, ferrous ion caused a small fluorescence quenching (89 - 97% of the initial fluorescence, over the range of iron tested) while ferric ion caused much greater quenching (65 - 88%). The most specific and linear response was observed for pyoverdin immobilized in sol-gel C. In contrast, a solution of pyoverdin (3.0 μM) exposed to iron (II or III) for 10 minutes showed an increase in fluorescence (101 - 114%) at low ferrous concentrations (0.45 - 2.18 μM) while exposure to all ferric ion concentrations (0.45 - 3.03 μM) caused quenching. In summary, the iron specificity of pyoverdin was improved by immobilizing it in sol-gel glass C.     

Binding and activity of all human alpha interferon subtypes

Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5–5 μM and 0.4–5 nM respectively (except for IFN-alpha1 – 220 nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC₅₀ values differ significantly (1.5 nM versus 0.1 nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.     

A dual-mode optical assay for iron(II) and gallic acid based on Fenton reaction

The hydroxyl radicals ( · OH) produced by the Fenton reaction of iron(II) and hydrogen peroxide (H₂O₂) can oxidize the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (Ox-TMB), resulting in a decrease in the fluorescence intensity of the reaction system and an increase in ultraviolet absorption. Ox-TMB had a visible absorption peak at 625 nm and a fluorescence peak around 420 nm. When gallic acid (GA) was added to the system, Ox-TMB was reduced to TMB, which made the color of the system disappear and the fluorescence recover. The linear ranges for determination of iron(II) were 0.5–10 μM (fluorometric) and 0.5–20 μM (colorimetric), and the detection limits were 0.25 μM (fluorometric) and 0.28 μM (colorimetric). The linear ranges for determination of GA were 0–80 μM (fluorometric) and 0–60 μM (colorimetric), and the detection limits were 0.31 μM (fluorometric) and 0.8 μM (colorimetric). The results of anti-interference experiments shew that this dual-mode assay had very good selectivity for the determination of iron(II) and GA.     

An improved labelling technique for monitoring microcrack growth in compact bone

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring.     

Synthesis and iron(III)-chelating properties of novel 3-hydroxypyridin-4-one hexadentate ligand-containing copolymers

Iron overload is a critical clinical problem that can be prevented by the use of iron-specific chelating agents. An alternative method of relieving iron overload is to reduce the efficiency of iron absorption from the intestine by administering iron chelators, which can bind iron irreversibly to form nontoxic, kinetically inert complexes that are not absorbed and are therefore excreted in the feces. A series of polymeric chelators with various iron binding capacities were therefore prepared as nonabsorbable iron-selective additives. A novel 3-hydroxypyridin-4-one hexadentate ligand CP254 has been synthesized and incorporated into polymers by copolymerisation with N, N-dimethylacrylamide (DMAA), and N, N'-ethylene-bis-acrylamide (EBAA) using (NH4)2S2O8 as the initiator. The physicochemical properties of CP254 were determined, namely, log K = 33.2 and pFe(3+) = 27.24. The chelating capacity of the CP254-DMAA copolymers was determined at physiological pH. The iron(III) chelation was found to achieve 80% capacity after 1 h and was virtually complete after 5 h, which is much quicker than that of the commercially available chelating resin Chelex100. The chelating copolymers were found to be readily regenerated and reusable. The copolymers possess a high selectivity for iron(III). The conditional affinity (log K') for iron(III) at pH 7.46 was determined to be 26.55, which is not significantly different to that of the hexadentate ligand CP254 (log K' = 26.47). In vitro perfusion studies indicate that the polymeric chelators described in this study can reduce iron absorption from the intestine.     

3-Aryl/Heteroaryl-5-amino-1-(3′,4′,5′-trimethoxybenzoyl)-1,2,4-triazoles as antimicrotubule agents. Design,synthesis, antiproliferative activity and inhibition of tubulin polymerization

Many natural and synthetic substances are known to interfere with the dynamic assembly of tubulin, preventing the formation of microtubules. In our search for potent and selective antitumor agents, a novel series of 1-(3′,4′,5′-trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had different heterocycles, including thiophene, furan or the three isomeric pyridines, and they possessed a phenyl ring bearing electron-releasing or electron-withdrawing substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-two tested compounds showed moderate to potent antiproliferative activities against a panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing strong antiproliferative activity (IC₅₀ < 1 μM) against selected cancer cells. Among them, several molecules preferentially inhibited the proliferation of leukemic cell lines, showing IC₅₀ values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly inhibited tubulin assembly, with an IC₅₀ value of 0.66 μM, half that obtained in simultaneous experiments with CA-4 (IC₅₀ = 1.3 μM).