Rita Santamaria

Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on β-amyloid-induced toxicity in PC12 cells

Alzheimers disease is widely held to be associated with oxidative stress due, in part, to the membrane action of β‐amyloid peptide aggregates. Here, we studied the effect of cannabidiol, a major non‐psychoactive component of the marijuana plant (Cannabis sativa) on β‐amyloid peptide‐induced toxicity in cultured rat pheocromocytoma PC12 cells. Following exposure of cells to β‐amyloid peptide (1 µg/mL), a marked reduction in cell survival was observed. This effect was associated with increased reactive oxygen species (ROS) production and lipid peroxidation, as well as caspase 3 (a key enzyme in the apoptosis cell‐signalling cascade) appearance, DNA fragmentation and increased intracellular calcium. Treatment of the cells with cannabidiol (10−7−10−4m) prior to β‐amyloid peptide exposure significantly elevated cell survival while it decreased ROS production, lipid peroxidation, caspase 3 levels, DNA fragmentation and intracellular calcium. Our results indicate that cannabidiol exerts a combination of neuroprotective, anti‐oxidative and anti‐apoptotic effects against β‐amyloid peptide toxicity, and that inhibition of caspase 3 appearance from its inactive precursor, pro‐caspase 3, by cannabidiol is involved in the signalling pathway for this neuroprotection.

Structure and cytotoxicity of phidianidines A and B: first finding of 1,2,4-oxadiazole system in a marine natural product.

Two indole alkaloids, phidianidines A (1) and B (2), exhibiting an uncommon 1,2,4-oxadiazole ring linked to the indole system, have been isolated from the marine opisthobranch mollusk Phidiana militaris. The structures of the two metabolites have been elucidated by spectroscopic techniques. Phidianidines exhibit high cytotoxicity against tumor and nontumor mammalian cell lines in in vitro assays.

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.

Cationic liposomes as efficient nanocarriers for the drug delivery of an anticancer cholesterol-based ruthenium complex

Aiming for novel tools for anticancer therapies, a ruthenium complex, covalently linked to a cholesterol-containing nucleolipid and stabilized by co-aggregation with a biocompatible lipid, is here presented. The amphiphilic ruthenium complex, named ToThyCholRu, is intrinsically negatively charged and has been inserted into liposomes formed by the cationic 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP) to hinder the degradation kinetics typically observed for known ruthenium-based antineoplastic agents. The here described nanovectors contain up to 30% in moles of the ruthenium complex and are stable for several weeks. This drug delivery system has been characterized using dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR) and electron paramagnetic resonance (EPR) techniques. Fluorescence microscopy, following the incorporation of rhodamine-B within the ruthenium-loaded liposomes, showed fast cellular uptake in human carcinoma cells, with a strong fluorescence accumulation within the cells. The in vitro bioactivity profile revealed an important antiproliferative activity and, most remarkably, the highest ability in ruthenium vectorization measured so far. Cellular morphological changes and DNA fragmentation provided evidence of an apoptosis-inducing activity, in line with several in vitro studies supporting apoptotic events as the main cause for the anticancer properties of ruthenium derivatives. Overall, these data highlighted the crucial role played by the cellular uptake properties in determining the anticancer efficacy of ruthenium-based drugs, showing DOTAP as a very efficient nanocarrier for their stabilization in aqueous media and transport in cells. In vitro bioscreens have shown the high antiproliferative activity of ToThyCholRu-DOTAP liposomes against specific human adenocarcinoma cell types. Furthermore, these formulations have proved to be over 20-fold more effective against MCF-7 and WiDr adenocarcinoma cells with respect to the nude ruthenium complex AziRu we have previously described.

Monocyte chemotactic protein-3 induces human coronary smooth muscle cell proliferation.

Monocyte chemotactic protein-3 (MCP-3), also known as CCL7, belongs to the monocyte chemotactic protein (MCP) subfamily of the CC chemokines that includes MCP-1/CCL2, MCP-2/CCL8, MCP-4/CCL13, and MCP-5/CCL12. Few studies have examined the role of MCP-3 in vascular pathologies such as atherosclerosis and restenosis in which smooth muscle cell (SMC) proliferation plays an important role. In this study, we investigated the effect of MCP-3 on human coronary artery smooth muscle cell (CASMC) proliferation. MCP-3 induced concentration-dependent CASMC proliferation with the maximum stimulatory effect at 0.3 ng/mL (about 50% vs unstimulated cells) assessed by bromodeoxyuridine (BrdU) uptake and direct cell counting. Anti-MCP-3 antibody (20 ng/mL) completely inhibited cell proliferation, demonstrating the specificity of the proliferative effect of MCP-3. Moreover, the MCP-3-induced CASMC proliferation was blocked by RS 102895 (0.06-6 μM), a specific antagonist of chemokine receptor 2 (CCR2). The mitogenic effect of MCP-3 appeared to be dependent on ERK1/2 MAPK and PI3K signaling pathway activation, as demonstrated by the reduction of MCP-3-induced CASMC proliferation observed after the treatment of cells with U0126 (1 μM) and LY-294002 (5μM), selective inhibitors of ERK 1/2 and PI3K activation, respectively. We found no relationship between MCP-3-induced CASMC proliferation and nuclear factor-κB activation. Moreover, we found that tumor necrosis factor-α (TNF-α, 30 ng/mL) and interleukin-1β (IL-1β, 1 ng/mL) both induced time-dependent increase of MCP-3 production by CASMCs, which was reduced by the anti-MCP-3 antibody (20 ng/mL), suggesting that the mitogenic effect of these stimuli is due, at least in part, to MCP-3. In conclusion, our results demonstrate that MCP-3 is produced by human CASMCs and directly induces CASMC proliferation in vitro, suggesting a potential role for this chemokine in vascular pathology.

Structural and functional analysis of aldolase B mutants related to hereditary fructose intolerance

Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired function of human liver aldolase (B isoform). 25 enzyme‐impairing mutations have been identified in the aldolase B gene. We have studied the HFI‐related mutant recombinant proteins W147R, A149P, A174D, L256P, N334K and Δ6ex6 in relation to aldolase B function and structure using kinetic assays and molecular graphics analysis. We found that these mutations affect aldolase B function by decreasing substrate affinity, maximal velocity and/or enzyme stability. Finally, the functional and structural analyses of the non‐natural mutant Q354E provide insight into the catalytic role of Arg303, whose natural mutants are associated to HFI.

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.

A new cytotoxic tambjamine alkaloid from the Azorean nudibranch Tambja ceutae

The chemical investigation of Azorean nudibranch mollusk Tambja ceutae led us to isolate a new member of the tambjamine family, tambjamine K (1). The bryozoan Bugula dentata, prey of the nudibranch, was also analyzed and found to contain compound 1 in very small amounts together with known blue pigment 2 and tambjamines A (3) and B (4). The structure of tambjamine 1 was elucidated by the interpretation of the spectroscopic data as well as by the comparison with related compounds. Compounds 1 and 2 possess antiproliferative activity, in particular, tambjamine K (1) displayed high cytotoxicity against both tumor and non-tumor mammalian cells.