Alfredo Colonna

Overexpression of H Ferritin and Up-regulation of Iron Regulatory Protein Genes during Differentiation of 3T3-L1 Pre-adipocytes

The role of iron-dependent oxidative metabolism in protecting the oxidable substrates contained in mature adipocytes is still unclear. Because differentiation increases ferritin formation in several cell types, thereby leading to an accumulation of H-rich isoferritins, we investigated whether differentiation affects iron metabolism in 3T3-L1 pre-adipocytes. To this aim, we evaluated the expression of the genes coding for the H and L ferritin subunits and for cytoplasmic iron regulatory protein (IRP) during the differentiation of 3T3-L1 cells in adipocytes induced by the addition of isobutylmethylxanthine, insulin, and dexamethasone. Differentiation enhanced ferritin formation and caused overexpression of the H subunit, thus altering the H/L subunit ratio. Northern blot analysis showed increased levels of H subunit mRNA. A gel retardation assay of cytoplasmic extract from differentiated cells, using an iron-responsive element as a probe, revealed enhanced an RNA binding capacity of IRP1, which correlated with the increase of IRP1 mRNA. The observed correlation between differentiation and iron metabolism in adipocytes suggests that an accumulation of H-rich isoferritin may limit the toxicity of iron in adipose tissue, thus exerting an antioxidant function.

Determination of pseudouridine and other nucleosides in human blood serum by high-performance liquid chromatography

A specific, sensitive, and rapid method to measure pseudouridine in human blood serum is described. The method is based on the following steps: (i) deproteinization of serum samples by filtration on membrane cones or by acetonitrile; (ii) purification of nucleosides and concentration of the sample by affinity chromatography on phenylboronate gel followed by lyophilization; and (iii) separation of nucleosides and their quantitation by reverse-phase high-performance liquid chromatography. The pseudouridine mean value in 30 normal subjects was 2.52 +/- 0.28 nmol/ml. The procedure also allows the identification of inosine, uridine, guanosine, and adenosine. Nevertheless, the presence in human blood serum of enzymatic activities which convert adenosine to inosine and cytidine to uridine prevents the precise quantitation of these nucleosides. All the compounds were identified by comparing their retention times and absorbance ratios (A280/A254) with those of pure compounds, as well as by cochromatography.

Cholesterol-Based Nucleolipid-Ruthenium Complex Stabilized by Lipid Aggregates for Antineoplastic Therapy

A novel ruthenium complex, linked to a cholesterol-containing nucleolipid (named ToThyCholRu), stabilized by lipid aggregates for antineoplastic therapy is presented. In order to retard the degradation kinetics typically observed for several ruthenium-based antineoplastic agents, ToThyCholRu is incorporated into a liposome bilayer formed by POPC. The resulting nanoaggregates contain up to 15% in moles of the ruthenium complex, and are shown to be stable for several weeks. The liposomes host the ruthenium-nucleolipid complex with the metal ion surrounded by POPC lipid headgroups and the steroid moiety inserted in the more external acyl chain region. These ruthenium-containing liposomes are more effective in inhibiting the growth of cancer cells than a model NAMI-A-like ruthenium complex, prepared for a direct evaluation of their anti-proliferative activity. These results introduce new perspectives in the design of innovative transition-metal-based supramolecular systems for anticancer drug vectorization.

High Fat Diet Induces Liver Steatosis and Early Dysregulation of Iron Metabolism in Rats

This paper is dedicated to the memory of our wonderful colleague Professor Alfredo Colonna, who passed away the same day of its acceptance. Fatty liver accumulation, inflammatory process and insulin resistance appear to be crucial in non-alcoholic fatty liver disease (NAFLD), nevertheless emerging findings pointed an important role also for iron overload. Here, we investigate the molecular mechanisms of hepatic iron metabolism in the onset of steatosis to understand whether its impairment could be an early event of liver inflammatory injury. Rats were fed with control diet or high fat diet (HFD) for 5 or 8 weeks, after which liver morphology, serum lipid profile, transaminases levels and hepatic iron content (HIC), were evaluated. In liver of HFD fed animals an increased time-dependent activity of iron regulatory protein 1 (IRP1) was evidenced, associated with the increase in transferrin receptor-1 (TfR1) expression and ferritin down-regulation. Moreover, ferroportin (FPN-1), the main protein involved in iron export, was down-regulated accordingly with hepcidin increase. These findings were indicative of an increased iron content into hepatocytes, which leads to an increase of harmful free-iron also related to the reduction of hepatic ferritin content. The progressive inflammatory damage was evidenced by the increase of hepatic TNF-α, IL-6 and leptin, in parallel to increased iron content and oxidative stress. The major finding that emerged of this study is the impairment of iron homeostasis in the ongoing and sustaining of liver steatosis, suggesting a strong link between iron metabolism unbalance, inflammatory damage and progression of disease.

Divergent modulation of iron regulatory proteins and ferritin biosynthesis by hypoxia/reoxygenation in neurons and glial cells

Ferritin, the main iron storage protein, exerts a cytoprotective effect against the iron‐catalyzed production of reactive oxygen species, but its role in brain injury caused by hypoxia/reoxygenation is unclear. Ferritin expression is regulated mainly at post‐transcriptional level by iron regulatory proteins (IRP1 and IRP2) that bind specific RNA sequences (IREs) in the 5′untranslated region of ferritin mRNA. Here, we show that hypoxia decreases IRP1 binding activity in glial cells and enhances it in cortical neurons. These effects were reversed by reoxygenation in both cell types. In glial cells there was an early increase of ferritin synthesis during hypoxia and reoxygenation. Conversely, in cortical neurons, ferritin synthesis increased during the late phase of reoxygenation. Steady‐state analysis of ferritin mRNA levels suggested that ferritin synthesis is regulated mainly post‐transcriptionally by IRPs in glioma cells, both transcriptionally and post‐transcriptionally in type‐1 astrocytes, and mainly at transcriptional level in an IRP‐independent way in neurons. The different regulation of ferritin expression may account for the different vulnerability of neurons and glial cells to the injury elicited by oxygen and glucose deprivation (OGD)/reoxygenation. The greater vulnerability of cortical neurons to hypoxia‐reoxygenation was strongly attenuated by the exogenous administration of ferritin during OGD/reoxygenation, suggesting the possible cytoprotective role exerted by this iron‐segregating protein.

Anticancer cationic ruthenium nanovectors: From rational molecular design to cellular uptake and bioactivity

An efficient drug delivery strategy is presented for novel anticancer amphiphilic ruthenium anionic complexes, based on the formation of stable nanoparticles with the cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP). This strategy is aimed at ensuring high ruthenium content within the formulation, long half-life in physiological media, and enhanced cell uptake. An in-depth microstructural characterization of the aggregates obtained mixing the ruthenium complex and the phospholipid carrier at 50/50 molar ratio is realized by combining a variety of techniques, including dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR), electron paramagnetic resonance (EPR), and zeta potential measurements. The in vitro bioactivity profile of the Ru-loaded nanoparticles is investigated on human and non-human cancer cell lines, showing IC(50) values in the low μM range against MCF-7 and WiDr cells, that is, proving to be 10-20-fold more active than AziRu, a previously synthesized NAMI-A analog, used for control. Fluorescence microscopy studies demonstrate that the amphiphilic Ru-complex/DOTAP formulations, added with rhodamine-B, are efficiently and rapidly incorporated in human MCF-7 breast adenocarcinoma cells. The intracellular fate of the amphiphilic Ru-complexes was investigated in the same in vitro model by means of an ad hoc designed fluorescently tagged analog, which exhibited a marked tendency to accumulate within or in proximity of the nuclei.

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.

Ovariectomy and estrogen treatment modulate iron metabolism in rat adipose tissue

Iron is essential for many biological processes and its deficiency or excess is involved in pathological conditions. At cellular level, the maintenance of iron homeostasis is largely accomplished by the transferrin receptor (TfR-1) and by ferritin, whose expression is mainly regulated post-transcriptionally by iron regulatory proteins (IRPs). This study examines the hypothesis that modification of serum estrogen levels by ovariectomy and 17beta-estradiol (E(2)) treatment in rats modulate serum iron-status parameters and iron metabolism in adipose tissue. In particular, we evaluated the RNA binding of IRP1 by electrophoretic mobility-shift assay and IRP1, ferritin, and TfR-1 expression in adipose tissue by Western blot analysis. Ovariectomy, besides a lowered serum iron and transferrin iron binding capacity, remarkably decreased the binding activity of IRP1 in peritoneal and subcutaneous adipose tissues, and these effects were reversed by E(2) treatment. Moreover, ovariectomy determined a decrease of IRP1 expression, which was significant in subcutaneous adipose tissue. Consistent with IRP1 regulation, an increase of ferritin and a decrease of TfR-1 expression were observed in peritoneal adipose tissue from ovariectomized animals, while the treatment with E(2) reconstituted TfR-1 level. A similar expression profile of TfR-1 was observed in subcutaneous adipose tissue, where ferritin level did not change in ovariectomized animals, and was increased after E(2) treatment. Our results indicate that estrogen level changes can regulate the binding activity of the IRP1, and consequently ferritin and TfR-1 expression in adipose tissue, suggesting a relationship among serum and tissue iron parameters, estrogen status and adiposity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells.

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Expression of iron-related proteins during infection by bovine herpes virus type-1.

Bovine herpesvirus 1 (BHV‐1), a dsDNA animal virus, is an economically important pathogen of cattle and the aetiological agent of many types of disease. The efficient replication of a DNA virus is strictly dependent on iron since this metal plays a crucial role in the catalytic center of viral ribonucleotide reductase. Consequently, iron metabolism is an important area for virus/host interaction and a large body of evidence suggests that viral infection is potentially influenced by the iron status of the host. The aim of the present study was to address the effects of BHV‐1 on iron metabolism in Madin‐Darby bovine kidney (MDBK) cells at different times of post‐infection. For this purpose, cell viability, iron regulatory proteins (IRPs) activity and levels, transferrin receptor 1 (TfR‐1), ferritin expression and LIP were evaluated. Our data demonstrate that a productive BHV‐1 infection in MDBK cells determines an overall decrease of IRPs RNA‐binding activity without affecting their expression. As consequence of this modulation, an increased ferritin mRNA translation and a decreased TfR‐1 mRNA translation were also observed. Moreover, the LIP level was decreased following viral infection. These results are consistent with the hypothesis that by reducing the iron up‐take and by enhancing the sequestration of free iron, animal cells will limit the iron availability for virus proliferation. Therefore, the results presented herein support the view that iron metabolism could be critical for the interaction between DNA viruses, such as BHV‐1, and mammalian cells. Delineation of the interplay among pathogen and host may provide new antimicrobial agents. J. Cell. Biochem. 104: 213–223, 2008.