Enzymatic spermine metabolites induce apoptosis associated with increase of p53, caspase-3 and mir-34a in both neuroblastoma cells, SJNKP and the N-Myc-amplified form IMR5

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down-or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis

Kinetics of rat CSD-C2 binding to H3.3 RNA

Cold-shock domain containing protein C2 (CSD-C2; also known as PIPPin) is an RNA-binding protein conserved in the evolution that interacts with the 3’-untranslated region (3’-UTR) of rat H1.0 and H3.3 histone messengers. Biolayer interferometry (BLI) is a technique that measures changes in an interference pattern generated from visible light, reflected from an optical layer, and a biolayer which contains molecules of interest. In this study, we used the BLI methodology in order to analyze and describe the binding properties of CSD-C2 and the mRNA encoding the rat brain histone protein H3.3. Recombinant CSD-C2 was incubated with in vitro transcribed, and biotinylated H3.3 RNA fragments bound to streptavidin-conjugated Octet optical biosensors. In order to define the RNA region involved in binding, we used RNA probes corresponding to different portions of H3.3 RNA 3’-UTR. In this study, we showed that CSD-C2 binds to the last 199 nucleotides of the H3.3 RNA 3’-UTR, and that the apparent affinity constant of the interaction is in the nanomolar range. In addition, this study confirmed that BLI can be a very efficient and reliable method for studying RNA-protein interactions

Toxic effects on astrocytes of extracellular vesicles from CSF of multiple sclerosis patients: A pilot in vitro study

Multiple sclerosis (MS) is an autoimmune and degenerative disorder of the central nervous system (CNS) that causes a progressive loss of motor and cognitive perfor-mances. Moreover, since the earlier phases, axonal loss as well as neuronal degener-ation and a failure of oligodendrocytes to promote myelin repair have been demon-strated. In previous studies, it has been shown that the treatment of rat neuronal primary cultures with serum from MS patients can be toxic for neurons. Here we report a pilot investigation showing that CSF from patients contains extracellular vesicles (EVs) able to induce cell death in rat cultured astrocytes. Although these data are still preliminary, they suggest at least two notable considerations: i) EVs can be instrumental to pathology, and their concentration in CSF might be pro-portional to MS severity; ii) astrocytes can be part of the degenerative process. As a consequence, we propose that cultured astrocytes can be used as a model to study the toxicity of EVs from patients affected by MS at different stages. In addition, we suggest that EVs and their cargoes might be used as biomarkers of MS severity