Molecular changes underlying decay of sensory responses and enhanced seizure propensity in peritumoral neurons

Background: Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target. Methods: We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection we analysed layer II-III pyramidal neurons molecular profile and with Local Field Potentials (LFP) recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice. Results: We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP-25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (i.e., AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signalling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in two mouse models of tumor-bearing mice. Conclusions: These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients' quality of life

Centrin 2: a novel marker of mature and neoplastic human astrocytes

As microtubule organizing centers, centrosomes play a pivotal role in cell division as well as in neurodevelopment and neuronal maturation. Among centrosomal proteins, centrin-2 (CETN2) contributes also to DNA repair mechanisms which are fundamental to prevent genomic instability during neural stem cell pool expansion. Nevertheless, the expression profile of CETN2 in human neural stem cells and their progeny is currently unknown. To address this question, we interrogated a platform of human neuroepithelial stem (NES) cells derived from post-mortem developing brain or established from pluripotent cells, and demonstrated that while CETN2 retains its centrosomal location in proliferating NES cells, its expression pattern changes upon differentiation. In particular, we found that CETN2 is selectively expressed in mature astrocytes with a broad cytoplasmic distribution. We then extended our findings on human autoptic nervous tissue samples. We investigated CETN2 distribution in diverse anatomical areas along the rostro-caudal neuraxis and pointed out a peculiar topography of CETN2-labelled astrocytes in humans which was not appreciable in murine tissues, where CETN2 was mostly confined to ependymal cells. As prototypical condition with glial overproliferation, we also explored CETN2 expression in glioblastoma multiforme, reporting a focal concentration of CETN2 in neoplastic astrocytes. This study expands CETN2 localization beyond centrosomes and reveals a unique expression pattern which makes it eligible as a novel astrocytic molecular marker, thus opening new roads to glial biology and human neural conditions

Complete Acid Ceramidase ablation prevents cancer-initiating cell formation in melanoma cells

Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma

Antioxidant activity and antiherpetic effects of a Solanum melongena L. genotype.

Herpes Simplex Virus type 1 (HSV-1) is a recurrent human virus, which develops quickly resistance to drugs commercially available, so increasing the need to study new sources of bioactive antiviral agents. To this end, extracts from medicinal plants, essential oils or fruits with antiviral properties are widely investigated in order to found the bioactive compounds. Among them, flavonoids and anthocyanins have been shown to inhibit the HSV-1, due to a probable virucidal action, likely antioxidant mechanisms (Khan et al., 2005). Besides, it is generally accepted that oxidative stress plays an important role in the pathogenesis of viral diseases (Peterhans, 1997). Also Solanaceae glycoalkaloids were found to be active against HSV-1 (Ikeda et al., 2000). On the basis of these evidences, in the present study, the antioxidant and antiherpetic properties of a DR2 eggplant (Solanum melongena L.) genotype (Mennella et al., 2012) were studied. Eggplant fruit is one of the most common vegetable consumed all around the world and an important source of both polyphenols and glycoalkaloids, including delphinidin, nasunin, chlorogenic acid and solamargine (Mennella et al., 2010). To perform the experiments, a 70% ethanol extract (pH 3) from the peel of the DR2 eggplant fruit, at both the commercial (B) and physiological (C) stage of ripeness, was prepared. The polyphenolic content was evaluated by high-performance thin-layer chromatography (HPTLC) and determined colorimetrically. Different antioxidant mechanisms, among which the radical scavenging power and the ability to block the ROS generation (by reducing and/or chelating mechanisms) were studied (Di Sotto et al., 2013). The antiherpetic activity of the extracts (DR2-B and DR2-C) was evaluated by the plaque assay in monkey kidney epithelial (Vero) cells, after infection with HSV-1 (Civitelli et al., 2014). In agreement with the colorimetric determinations, the HPTLC analysis showed the presence of different polyphenols in both the extracts, particularly the anthocyanin, delphinidin 3-O-β-rutinoside. The samples possessed antioxidant properties, being able to scavenge different radical species and to block the ROS generation by chelating mechanisms. As regard the antiherpetic activity, in spite of a null effect of DR2-B, the extract DR2-C inhibited the HSV-1 replication in a dose-dependent manner, reaching a 93% inhibition at concentration of 500 g/ml. When administered during different phases of the virus life-cycle, DR2-C inhibited the viral replication of about 50% during the adsorption period: these data were confirmed by the immunoblotting analysis, in which several herpetic proteins resulted inhibited. Present data highlight that DR2-C extract possess antiherpetic properties, likely due to an impairment of specific steps of the virus life-cycle. Taking into account that the HSV-1 replication requires an impairment of the intracellular redox status, the antioxidant properties of DR2-C extract, likely due to the presence of different polyphenolic compounds, could be involved in the antiviral effects found. In conclusion, the beneficial antioxidant and antiherpetic properties of DR-2C suggest a possible application of S. melongena as dietary supplement, or included in topical formulations, to treat the herpetic skin symptomatic lesions

Mouse mammary tumour virus-like env nucleotide and p14 signal peptide are present in feline mammary carcinomas, but not in neoplastic or dysplastic canine mammary lesions

Mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer and it has been hypothesized that companion animals might have a role in viral transmission. The aim of our study was to investigate the presence of MMTV-like nucleotide sequences and viral protein in a larger number of feline (FMCs) and canine mammary carcinomas (CMCs) by nested PCR and immunohistochemistry. Results showed that the presence of MMTV-like env sequence in FMCs was 7% (6/86), while all the CMCs and canine dysplastic lesions scored negative. All PCR-positive FMCs scored positive for the MMTV p14 signal peptide of the envelope precursor protein of the virus. In contrast, all PCR-negative FMCs and canine mammary lesions were also negative for immunohistochemistry analysis. Canine and feline normal mammary gland tissues scored negative for both PCR and MMTV-p14 protein. Multiple nucleotide alignment of MMTV-like env gene sequences isolated from cat showed 97% and 99% similarity with HMTV and MMTV, respectively, while the others two presented some polimorphisms. Particularly the sequences of one of these two tumors showed a polymorphism (c.7575 A> G), that causes a previously unreported amino acid substitution (Thr > Ala). In conclusion, the results of our study showed the presence of MMTV-like sequences and viral protein in some FMCs. Further studies are needed to understand whether this virus does play a role in the development of FMCs, if MMTV-like is an exogenous virus as these data suggest and, in such a case, how and from whom this virus was acquired

The SUCCESSO-TERRA Project: a Lesson of Sustainability from the Terramare Culture, Middle Bronze Age of the Po Plain (Northern Italy)

This backstory article deals with the SUCCESSO-TERRA Project (2017–2020), an interdisciplinary research program aiming at reconstructing the land-use transformations that occurred during the development of the Terramare culture in the southern-central Po Plain of Northern Italy. Topics include climate-environment changes, human impact and exploitation of natural resources that are interconnected topics in human ecology and environmental sciences. These topics can only be understood in a long-term perspective integrating archaeology, geology, botany and other sciences. The text includes the theoretical basis, the research strategy and the main methodological approaches given by geoarchaeology and palynology, the two research sides constituting the partnership of the project

A human MMTV-like betaretrovirus linked to breast cancer has been present in humans at least since the copper age

The betaretrovirus Mouse Mammary Tumor Virus (MMTV) is the well characterized etiological agent of mammary tumors in mice. In contrast, the etiology of sporadic human breast cancer (BC) is unknown, but accumulating data indicate a possible viral origin also for these malignancies. The presence of MMTVenv-like sequences (MMTVels) in the human salivary glands and saliva supports the latter as possible route of interhuman dissemination. In the absence of the demonstration of a mouse-man transmission of MMTV, we considered the possibility that a cross-species transmission could have occurred in ancient times. Therefore, we investigated MMTVels in the ancient dental calculus, which originates from saliva and is an excellent material for paleovirology. The calculus was collected from 36 ancient human skulls, excluding any possible mouse contamination. MMTV-like sequences were identified in the calculus of 6 individuals dated from the Copper Age to the 17th century. The MMTV-like sequences were compared with known human endogenous betaretroviruses and with animal exogenous betaretroviruses, confirming their exogenous origin and relation to MMTV. These data reveal that a human exogenous betaretrovirus similar to MMTV has existed at least since 4,500 years ago and indirectly support the hypothesis that it could play a role in human breast cancer

Inhibition of chloride intracellular channel 1 (CLIC1) as biguanide class-effect to impair human glioblastoma stem cell viability

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC50values significantly lower than metformin, (range 0.054-0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanidestructured molecules, characterized by high antitumor efficacy and safe toxicological profile

A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: Murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers

Multiregional sequencing of IDH-WT glioblastoma reveals high genetic heterogeneity and a dynamic evolutionary history

Glioblastoma is one of the most common and lethal primary neoplasms of the brain. Patient survival has not improved significantly over the past three decades and the patient median survival is just over one year. Tumor heterogeneity is thought to be a major determinant of therapeutic failure and a major reason for poor overall survival. This work aims to comprehensively define intra- and inter-tumor heterogeneity by mapping the genomic and mutational landscape of multiple areas of three primary IDH wild-type (IDH-WT) glioblastomas. Using whole exome sequencing, we explored how copy number variation, chromosomal and single loci amplifications/deletions, and mutational burden are spatially distributed across nine different tumor regions. The results show that all tumors exhibit a different signature despite the same diagnosis. Above all, a high inter-tumor heterogeneity emerges. The evolutionary dynamics of all identified mutations within each region underline the questionable value of a single biopsy and thus the therapeutic approach for the patient. Multiregional collection and subsequent sequencing are essential to try to address the clinical challenge of precision medicine. Especially in glioblastoma, this approach could provide powerful support to pathologists and oncologists in evaluating the diagnosis and defining the best treatment option