Adriana Carol Eleonora Graziano

An engineered nanoplatform for bimodal anticancer phototherapy with dual-color fluorescence detection of sensitizers

A multifunctional nanoplatform with four-in-one photoresponsive functionalities has been achieved through the co-encapsulation of two chromo-fluorogenic components within biocompatible polymeric nanoparticles. This engineered nanoconstruct efficiently delivers different photosensitizers in melanoma cells, which can be detected through their dual-color fluorescence, and induces amplified cell mortality due to the simultaneous photogeneration of singlet oxygen and nitric oxide.

Sodium L-Lactate Differently Affects Brain-Derived Neurothrophic Factor, Inducible Nitric Oxide Synthase, and Heat Shock Protein 70 kDa Production in Human Astrocytes and SH-SY5Y Cultures

The present study analyzed the in vitro effects induced by sodium L‐lactate on human astrocytes and the SH‐SY5Y cell line, when added at concentrations of 5, 10, and 25 mmol/liter. Expression of brain‐derived neurotrophic factor (BDNF), inducible nitric oxide synthase (iNOS), and heat shock protein 70 kDa (HSP70) was evaluated by Western blot analysis. Cell viability with MTT, release of nitric oxide (NO) through the Griess reaction, and production of BDNF by enzyme‐linked immunoassay was determined. Data indicate that, in SH‐SY5Y as well as in cortical astrocytes, after 4 hr sodium L‐lactate increases the expression and release of BDNF, iNOS, and NO; after 24 hr, it turns is ineffective for the production of the neurotrophin in SH‐SY5Y and not in astrocytes, but the expression of iNOS and release of NO appear to be further increased compared with those after 4 hr. Sodium L‐lactate influences differently the expression of HSP70 in SH‐SY5Y compared with astrocytes. We propose, based on these findings, that sodium L‐lactate affects the expression of BDNF in SH‐SY5Y and astrocytes in a different manner: high levels of iNOS and NO expressed in SH‐SY5Y have a profound inhibitory effect on the release of BDNF related to a more limited production of HSP70 by SH‐SY5Y. In conclusion, the results demonstrate differences in the responses of SH‐SY5Y and astrocytes to stimulation by high levels of sodium L‐lactate. Sodium L‐lactate differently and dose and time dependently influences the expression and release of BDNF, iNOS, NO, and HSP70 depending on the cell type.

Photoinduced Fluorescence Activation and Nitric Oxide Release with Biocompatible Polymer Nanoparticles

A viable strategy to encapsulate a fluorophore/photochrome dyad and a nitric oxide photodonor within supramolecular assemblies of a cyclodextrin-based polymer in water was developed. The two photoresponsive guests do not interact with each other within their supramolecular container and can be operated in parallel under optical control. Specifically, the dyad permits the reversible switching of fluorescence on a microsecond timescale for hundreds of cycles, and the photodonor enables the irreversible release of nitric oxide. Furthermore, these supramolecular assemblies cross the membrane of human melanoma cancer cells and transport their cargo in the cytosol. The fluorescence of one component allows the visualization of the labeled cells, and its switchable character could, in principle, be used to acquire super-resolution images, while the release of nitric oxide from the other induces significant cell mortality. Thus, our design logic for the construction of biocompatible nanoparticles with dual functionality might evolve into the realization of valuable photoresponsive probes for imaging and therapeutic applications.

Krabbe disease: involvement of connexin43 in the apoptotic effects of sphingolipid psychosine on mouse oligodendrocyte precursors

Krabbe disease is a genetic demyelinating syndrome characterized by deficiency of the enzyme β-galactosylceramidase, lysosomal psychosine accumulation, and loss of myelin-forming cells. In this study, some apoptotic markers such as apoptotic index (AI), DNA fragmentation, caspase-3, PTEN, Bad, and PI3K were determined in oligodendrocyte precursors from wild type or twitcher mice untreated or treated with psychosine. Twitcher is a natural mouse model of Krabbe disease containing a premature stop codon (W339X) in the β-galactosylceramidase gene. Moreover, a possible involvement of connexin (Cx)43 in cell death of oligodendrocyte precursors induced by psychosine was investigated with the final aim to provide a contribution to the knowledge of the molecular mechanisms and pathophysiological events that occur in Krabbe disease. Connexins are a multigene family of structurally related trans-membrane proteins able to modulate essential cellular processes such as proliferation, differentiation and migration. Among these, Cx43 is the predominant isoform in many cell types, including neural progenitor cells. Our results showed an increase of AI, DNA fragmentation, caspase-3, PTEN, Bad, and Cx43 associated to a decrease of PI3K, pAKT and pBad. Taken together, these findings suggest an involvement of Cx43 in the psychosine-mediated apoptosis of primary oligodendrocyte progenitors from wild type or twitcher mice, used for the first time as cell models in comparison. It could open unexplored perspective also for other demyelinating diseases.

A Citrus bergamia Extract Decreases Adipogenesis and Increases Lipolysis by Modulating PPAR Levels in Mesenchymal Stem Cells from Human Adipose Tissue

The aim of this research was to assess the impact of a well-characterized extract from Citrus bergamia juice on adipogenesis and/or lipolysis using mesenchymal stem cells from human adipose tissue as a cell model. To evaluate the effects on adipogenesis, some cell cultures were treated with adipogenic medium plus 10 or 100 μg/mL of extract. To determine the properties on lipolysis, additional mesenchymal stem cells were cultured with adipogenic medium for 14 days and after this time added with Citrus bergamia for further 14 days. To verify adipogenic differentiation, oil red O staining at 7, 14, 21, and 28 days was performed. Moreover, the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ), adipocytes fatty acid-binding protein (A-FABP), adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), monoglyceride lipase (MGL), 5′-adenosine monophosphate-activated protein kinase (AMPK)α1/2, and pAMPKα1/2 was evaluated by Western blot analysis and the release of glycerol by colorimetric assay. Citrus bergamia extract suppressed the accumulation of intracellular lipids in mesenchymal stem cells during adipogenic differentiation and promoted lipolysis by repressing the expression of adipogenic genes and activating lipolytic genes. Citrus bergamia extract could be a useful natural product for improving adipose mobilization in obesity-related disorders.

Light-Tunable Generation of Singlet Oxygen and Nitric Oxide with a Bichromophoric Molecular Hybrid: a Bimodal Approach to Killing Cancer Cells.

The design, synthesis, photochemical properties, and biological evaluation of a novel photoactivatable bichromophoric conjugate are reported. The compound 1, [4‐(4,4‐difluoro‐2,6‐diiodo‐1,3,5,7‐tetramethyl‐4‐bora‐3a,4a‐diaza‐s‐indacen‐8‐yl)‐N‐(3‐((4‐nitro‐3‐(trifluoromethyl)phenyl)amino)propyl)butanamide] combines a 2,6‐diiodo‐1,3,5,7‐tetramethyl BODIPY derivative as singlet oxygen (1O2) photosensitizer and 4‐nitro‐3‐(trifluoromethyl)aniline (NOPD) as nitric oxide (NO) photodonor, joined by an alkyl spacer. These two chromogenic units absorb in distinct regions of the visible spectrum, and their individual photochemical properties are conserved in the molecular conjugate. Irradiation of the bichromophoric conjugate with green light afforded 1O2 in high quantum yields, whereas 1O2 production was negligible with the use of blue light; under this latter condition, NO was released. Photogeneration of NO and cytotoxic 1O2 can therefore be regulated by appropriately tuning the excitation light wavelength and intensity. Tested on melanoma cancer cells, this resulted in amplified photomortality relative to that of a structurally correlated model compound 2 [4‐(4,4‐difluoro‐2,6‐diiodo‐1,3,5,7‐tetramethyl‐4‐bora‐3a,4a‐diaza‐s‐indacen‐8‐yl)‐N‐(3‐(p‐tolylamino)propyl)butanamide] deprived of the NO‐release capacity. The cellular uptake of 1, evaluated by confocal fluorescence microscopy, showed that the product is localized in the cytoplasm.

Wilms' tumor gene 1 (WT1) silencing inhibits proliferation of malignant peripheral nerve sheath tumor sNF96.2 cell line.

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.

Differentiation of human adipose stem cells into neural phenotype by neuroblastoma‐ or olfactory ensheathing cells‐conditioned medium

Olfactory ensheathing cells (OECs) are known to be capable of continuous neurogenesis throughout lifetime and are a source of multiple trophic factors important in central nervous system regeneration. B104 neuroblastoma cells are recognized to induce differentiation of neural stem cells into oligodendrocyte precursor cells. Therefore, the aim of this study was to verify if conditioned medium (CM) obtained from OECs or B104 cells was capable of inducing differentiation of adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) to a neuronal phenotype. In order to this goal, immunocytochemical procedures and flow cytometry analysis were used and some neural markers, as nestin, protein gene product 9.5 (PGP 9.5), microtubule‐associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and neuron cell surface antigen (A2B5) were examined 24 h and 7 days after the treatment. The results showed that both OECs‐ or B104‐CM treated AT‐MSCs express markers of progenitor and mature neurons (nestin, PGP 9.5 and MAP2) in time‐dependent manner, display morphological features resembling neuronal cells, and result negative for GFAP and A2B5, astrocyte and oligodendrocyte markers, respectively. This study demonstrated that AT‐MSCs can be influenced by the environment, indicating that these cells can respond to environmental cues also versus a neuronal phenotype. J. Cell. Physiol. 228: 2109–2118, 2013.

A Multifunctional Bichromophoric Nanoaggregate for Fluorescence Imaging and Simultaneous Photogeneration of RNOS and ROS

We report the design, preparation, and properties of a nanoaggregate formed in phosphate buffer solution by a porphyrin-β-cyclodextrin (β-CD) conjugate and a nitric oxide photodonor tailored to fit the β-CD cavity. The small nanoassembly with a diameter of about 13 nm exhibits the typical red fluorescence of the porphyrin chromophore. The empty cavity of the β-CD unit in the nanoaggregate is able to accommodate the NO photodonor, thereby forming a supramolecular bichromophoric aggregate with diameter of about 16 nm. This nanoconstruct preserves the fluorescence of the porphyrin core and is able to generate nitric oxide and singlet oxygen under illumination with visible light. The nanoassembly internalizes in melanoma cells, can be mapped therein by fluorescence microscopy, and induces a significant level of cell mortality, probably due to the combined action of reactive nitrogen oxide species (RNOS) and reactive oxygen species (ROS).

A multi-photoresponsive molecular-hybrid for dual-modal photoinactivation of cancer cells

We report the design, synthesis, photochemical characterization and biological evaluation of a novel molecular conjugate in which two chromogenic centers, a porphyrin unit and a nitroaniline derivative, are covalently linked through an alkyl spacer. This molecular hybrid can be encapsulated in biocompatible, water soluble polymer nanoparticles where it shows satisfactory fluorescence emission and capability to generate simultaneously the cytotoxic singlet oxygen and nitric oxide upon excitation with visible light. The photoactive nanoassembly can be delivered to A375 melanoma cancer cells where it can be detected through its red fluorescence, and is capable of inducing amplified cell mortality by bimodal action due to the concomitant photoproduction of reactive oxygen and nitrogen oxygen species.