Panagiota Papazafiri

Anti-apoptotic effects of allopregnanolone on P19 neurons

Progesterone and its metabolites are potent allosteric modulators of GABAA receptor function, through a direct, non‐genomic interaction with specific receptor subtypes. In addition, fluctuations in the concentration of progesterone, and allopregnanolone in particular, have been shown to modulate GABAA receptor gene expression and activity. In this study, mouse P19 cells were induced to differentiate into post‐mitotic neurons which express specific neuronal markers, including GABAA and N‐methyl‐d‐aspartate (NMDA) receptors. Apoptotic cell death, induced in the presence of NMDA, was efficiently prevented by allopregnanolone and dehydroepiandrosterone (DHEA) but not DHEA sulfate. Apoptosis was accompanied by cytochrome c release to the cytoplasm and Bax translocation to the mitochondria, while the levels of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL remained unchanged. In the presence of the most potent neurosteroid, allopregnanolone, DNA fragmentation as well as cytochrome c and Bax translocation were prevented. On the other hand, short‐term exposure (1–20 µm, 24 h) of P19‐derived neurons to allopregnanolone and DHEA significantly increased the levels of α1 and β2 mRNAs of GABAA receptor, while the levels of NR1 mRNA of NMDA receptor were not altered. These results suggest that neurosteroids, interfering with the mitochondrial apoptotic pathway, are able to act as survival factors in neuronal cells, while they contribute to GABAA receptor plasticity modulating the expression of its subunits.

Cytotoxic halogenated metabolites from the Brazilian red alga Laurencia catarinensis.

Seven new (1-7) and seven previously reported (8-14) halogenated metabolites were isolated from the organic extract of the Brazilian red alga Laurencia catarinensis. The structure elucidation and the assignment of the relative configurations of the new natural products were based on detailed NMR and MS spectroscopic analyses, whereas the structure of metabolite 6 was confirmed by single-crystal X-ray diffraction analysis. The absolute configuration of metabolite 1 was determined using the modified Moshers method. The in vitro cytotoxicity of compounds 1-14 was evaluated against HT29, MCF7, and A431 cell lines.

Deregulation of calcium homeostasis mediates secreted α–synuclein-induced neurotoxicity

α-Synuclein (AS) plays a crucial role in Parkinsons disease pathogenesis. AS is normally secreted from neuronal cells and can thus exert paracrine effects. We have previously demonstrated that naturally secreted AS species, derived from SH-SY5Y cells inducibly overexpressing human wild type AS, can be toxic to recipient neuronal cells. In the current study, we show that application of secreted AS alters membrane fluidity and increases calcium (Ca2+) entry. This influx is reduced on pharmacological inhibition of voltage-operated Ca2+ channels. Although no change in free cytosolic Ca2+ levels is observed, a significantly increased mitochondrial Ca2+ sequestration is found in recipient cells. Application of voltage-operated Ca2+ channel blockers or Ca2+ chelators abolishes AS-mediated toxicity. AS-treated cells exhibit increased calpain activation, and calpain inhibition greatly alleviates the observed toxicity. Collectively, our data suggest that secreted AS exerts toxicity through engagement, at least in part, of the Ca2+ homeostatic machinery. Therefore, manipulating Ca2+ signaling pathways might represent a potential therapeutic strategy for Parkinsons disease.

Towards an alternative testing strategy for nanomaterials used in nanomedicine: Lessons from NanoTEST

Abstract In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project (www.nanotest-fp7.eu) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

Attenuation of oxidative stress in HL-1 cardiomyocytes improves mitochondrial function and stabilizes Hif-1α

HL-1 cardiomyocytes were subjected to simulated hypoxia, in the presence of cobalt chloride, which resulted in reduction of cell viability and induction of DNA laddering, indicating the activation of the apoptotic cascade. In the presence of trolox, ascorbic acid, melatonin and the hybrid compound of trolox and lipoic acid (LaT 3a), cell viability was increased, with LaT 3a exhibiting the best effect. Antioxidant treatment restored ATP levels, abolished laddering of DNA, abrogated MPTP opening, Bax translocation to the mitochondria and cytochrome c release to the cytoplasm. Moreover, severe hypoxia, was found to destabilize hypoxia inducible factor-1α (Hif-1α) mRNA. Reduction of oxidative stress attenuated this effect, implying a possible anti-apoptotic action of the master regulator of hypoxia response. Our data suggest that antioxidants can maintain cell function and survival by inhibiting the mitochondrial apoptotic pathway and stabilizing Hif-1α.

Tetrahydrofuran Acetogenins from Laurencia glandulifera

Five new C(15) acetogenin en-ynes (1-5) with a rare tetrahydrofuran moiety and a linear biosynthetic precursor (6) were isolated from an organic extract of Laurencia glandulifera, collected from the island of Crete in the south Aegean Sea. The structures of the new natural products, as well as their relative configuration, were established by means of spectroscopic data analysis. The cytotoxicity of the isolated natural products was evaluated against five human tumor cell lines.

Pharmacophore modeling for qualitative prediction of antiestrogenic activity.

A ligand-based pharmacophore approach for the prediction of antiestrogenic activity to be used as an in silico screening tool for bioactive compounds including natural products was developed using Catalyst HypoGen. The generated pharmacophore hypothesis (HYPO-7) consisted of five features, namely, one hydrophobic (HY1), two hydrophobic aromatic (HY2), one hydrogen-bond acceptor (HBA), and one hydrogen-bond donor (HBD). HYPO-7 successfully predicted the lack of cytotoxicity of a number of new metabolites isolated from the red alga Laurencia glandulifera. Furthermore, a screening of the Asinex Gold Collection database was performed by coupling HYPO-7 with a docking filtration, which resulted in a restricted set of 12 new scaffolds to be investigated as potential SERMs. The inhibitory activity of these compounds was evaluated in vitro using MCF7 human breast adenocarcinoma cell line. Ten out of the twelve compounds exhibited inhibitory activity with IC(50) values between 26 and 188 microM. This result shows that application of HYPO-7 could assist in the selection of potentially active compounds, thus expediting the hit discovery process.

Effect of nanostructured TiO2 crystal phase on photoinduced apoptosis of breast cancer epithelial cells

Purpose The use of nanoparticles has seen exponential growth in the area of health care, due to the unique physicochemical properties of nanomaterials that make them desirable for medical applications. The aim of this study was to examine the effects of crystal phase-nanostructured titanium dioxide particles on bioactivity/cytotoxicity in breast cancer epithelial cells. Materials and methods Cultured Michigan Cancer Foundation (MCF)-7 and human breast adenocarcinoma (MDA-MB-468) breast cancer epithelial cells were exposed to ultraviolet A light (wavelength 350 nm) for 20 minutes in the presence of aqueous dispersions of two different nanostructured titanium dioxide (TiO2) crystal phases: anatase and an anatase–rutile mixture. Detailed characterization of each titanium dispersion was performed by dynamic light scattering. A 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) colorimetric assay was employed to estimate the percentage of viable cells after each treatment. Western blot analysis of protein expression and characterization, as well as a deoxyribonucleic acid (DNA)-laddering assay, were used to detect cell apoptosis. Results Our results documented that 100% anatase TiO2 nanoparticles (110–130 nm) exhibited significantly higher cytotoxicity in the highly malignant MDA-MB-468 cancer cells than anatase– rutile mixtures (75%/25%) with the same size. On the contrary, MCF-7 cells (characterized by low invasive properties) were not considerably affected. Exposure of MDA-MB-468 cells to pure anatase nanoparticles or anatase–rutile mixtures for 48 hours resulted in increased proapoptotic Bax expression, caspase-mediated poly(adenosine diphosphate ribose) polymerase (PARP) cleavage, DNA fragmentation, and programmed cell death/apoptosis. Conclusion The obtained results indicated that pure anatase TiO2 nanoparticles exhibit superior cytotoxic effects compared to anatase–rutile mixtures of the same size. The molecular mechanism of TiO2 nanoparticle cytotoxicity involved increased Bax expression and caspase-mediated PARP inactivation, thus resulting in DNA fragmentation and cell apoptosis.

Neuroprotective effects of steroid analogues on P19-N neurons.

Naturally occurring neurosteroids are potent allosteric modulators of gamma-aminobutyric acid(A) receptor and through augmentation of gamma-aminobutyric acid(A) receptor function, can protect neuronal cells against N-methyl-d-aspartate receptor over-activation, ischemia and traumatic brain injury. In this study, mouse P19 cells were induced to differentiate into post-mitotic neurons and were subjected to excitotoxicity in the presence of N-methyl-d-aspartate. Novel synthetic analogues of the endogenous neurosteroids allopregnanolone and dehydroepiandrostrone, inhibited excitotoxic cell death of P19-N neurons, by directly maintaining the activation of PKB/Akt kinase and interfering with the intrinsic mitochondrial apoptotic pathway, preserving cytochrome c in the mitochondria and Bax in the cytoplasm. The efficiency and the potency of these neurosteroids were similar to those of allopregnanolone and dehydroepiandrostrone. Their effects were gamma-aminobutyric acid(A) receptor mediated, since they were abolished in the presence of bicuculline, an antagonist of receptors function. In addition, the synthetic compounds retained the ability to alter gamma-aminobutyric acid(A) receptor subunit gene expression, but their effects on transcriptional activity were less pronounced than those of allopregnanolone and dehydroepiandrostrone. These results suggest that synthetic neurosteroids may serve as potent, membrane acting, neuroprotectants against N-methyl-d-aspartate receptor neurotoxicity on neuronal cells.

Developmental and age-related alterations of calcium homeostasis in human fibroblasts.

Calcium homeostasis, in terms of both cytosolic calcium concentration ([Ca(2+)](i)) and capacitative calcium entry, has been investigated in human skin and lung fibroblasts at different developmental and ageing stages by employing the Fura-2 based measurements. [Ca(2+)](i) in foetal lung or skin cells were nearly similar. However, significant changes were observed between foetal and adult fibroblasts and interestingly in opposite directions depending on the tissue of origin. In particular, in adult lung cells [Ca(2+)](i) was more than three-fold higher compared to adult skin fibroblasts, a difference which may contribute to tissue-specific functions. Capacitative Ca(2+) entry, i.e. the transient [Ca(2+)](i) increase induced by re-addition of extracellular calcium after depletion of thapsigargin-sensitive calcium stores, was found to exhibit the same pattern of differences during foetal-to-adult transition (it is two-fold higher in adult lung cells than in adult skin cells). At variance, we found capacitative Ca(2+) entry to be significantly attenuated during in vivo or in vitro ageing of fibroblasts; while minor alterations of [Ca(2+)](i) were observed. These findings indicate that capacitative calcium entry rather than [Ca(2+)](i), is mainly affected during the ageing process.