Serena Vella

Selection of candidate housekeeping genes for normalization in human postmortem brain samples.

The most frequently used technique to study the expression profile of genes involved in common neurological disorders is quantitative real-time RT-PCR, which allows the indirect detection of very low amounts of selected mRNAs in tissue samples. Expression analysis by RT-qPCR requires an appropriate normalization to the expression level of genes characterized by a stable, constitutive transcription. However, the identification of a gene transcribed at a very stable level is difficult if not impossible, since significant fluctuations of the level of mRNA synthesis often accompanies changes of cell behavior. The aim of this study is to identify the most stable genes in postmortem human brain samples of patients affected by Alzheimer’s disease (AD) suitable as reference genes. The experiments analyzed 12 commonly used reference genes in brain samples from eight individuals with AD and seven controls. After a careful analysis of the results calculated by geNorm and NormFinder algorithms, we found that CYC1 and EIF4A2 are the best reference genes. We remark on the importance of the determination of the best reference genes for each sample to be analyzed and suggest a practical combination of reference genes to be used in the analysis of human postmortem samples.

Dichloroacetate inhibits neuroblastoma growth by specifically acting against malignant undifferentiated cells

The small, water soluble molecule Dichloroacetate (DCA) is recently arousing lively interests in the field of cancer therapy for it has been shown to be able to inhibit the growth of human tumors acting specifically on the mitochondria of cancer cells without perturbing the physiology of nonmalignant cells. Neuroblastoma was one of the tumor types on which DCA was considered ineffective as it is composed of cells with few recognized mitochondrial anomalies. Neuroblastoma, however, is composed of different cell types in terms of metabolism, phenotype and malignant potential. Despite the above prediction, in this work, we show that (i) DCA exhibits an unexpected anticancer effect on NB tumor cells and (ii) this effect is selectively directed to very malignant NB cells, whereas the more differentiated/less malignant NB cells are refractory to DCA treatment. This result supports the need of a detailed investigation of DCA anticancer properties against this tumor type with the final aim of its possible use as therapeutic agent.

NDM29, a RNA polymerase III-dependent non coding RNA, promotes amyloidogenic processing of APP and amyloid β secretion.

Neuroblastoma Differentiation Marker 29 (NDM29) is a RNA polymerase (pol) III-transcribed non-coding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype. Since in this process a complex pattern of molecular changes is associated to plasma membrane protein repertoire we hypothesized that the expression of NDM29 might influence also key players of neurodegenerative pathways. In this work we show that the NDM29-dependent cell maturation induces amyloid precursor protein (APP) synthesis, leading to the increase of amyloid β peptide (Aβ) secretion and the concomitant increment of Aβ x-42/Aβ x-40 ratio. We also demonstrate that the expression of NDM29 RNA, and the consequent increase of Aβ formation, can be promoted by inflammatory stimuli (and repressed by anti-inflammatory drugs). Moreover, NDM29 expression was detected in normal human brains although an abnormal increased synthesis of this ncRNA is induced in patients affected by neurodegenerative diseases. Therefore, the complex of events triggered by NDM29 expression induces a condition that favors the formation of Aβ peptides in the extracellular space, as it may occur in Alzheimers Disease (AD). In addition, these data unexpectedly show that a pol III-dependent small RNA can act as key regulator of brain physiology and/or pathology suggesting that a better knowledge of this portion of the human transcriptome might provide hints for neurodegeneration studies.

Advances in emerging drugs for osteosarcoma

Introduction: Osteosarcoma (OS), the most common primary malignant bone tumor, is currently treated with pre- and postoperative chemotherapy in association with the surgical removal of the tumor. Conventional treatments allow to cure about 60 – 65% of patients with primary tumors and only 20 – 25% of patients with recurrent disease. New treatment approaches and drugs are therefore highly warranted to improve prognosis. Areas covered: This review focuses on the therapeutic approaches that are under development or clinical evaluation in OS. Information was obtained from different and continuously updated data bases, as well as from literature searches, in which particular relevance was given to reports and reviews on new targeted therapies under clinical investigation in high-grade OS. Expert opinion: OS is a heterogeneous tumor, with a great variability in treatment response between patients. It is therefore unlikely that a single therapeutic tool will be uniformly successful for all OS patients. This claims for the validation of new treatment approaches together with biologic/(pharmaco)genetic markers, which may select the most appropriate subgroup of patients for each treatment approach. Since some promising novel agents and treatment strategies are currently tested in Phase I/II/III clinical trials, we may hope that new therapies with superior efficacy and safety profiles will be identified in the next few years.

EZH2 Down-Regulation Exacerbates Lipid Accumulation and Inflammation in in Vitro and in Vivo NAFLD

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent, chronic liver diseases, worldwide. It is a multifactorial disease caused by complex interactions between genetic, epigenetic and environmental factors. Recently, several microRNAs, some of which epigenetically regulated, have been found to be up- and/or down-regulated during NAFLD development. However, in NAFLD, the essential role of the Polycomb Group protein Enhancer of Zeste Homolog 2 (EZH2), which controls the epigenetic silencing of specific genes and/or microRNAs by trimethylating Lys27 on histone H3, still remains unknown. In this study, we demonstrate that the nuclear expression/activity of the EZH2 protein is down-regulated both in livers from NAFLD rats and in the free fatty acid-treated HepG2. The drop in EZH2 is inversely correlated with: (i) lipid accumulation; (ii) the expression of pro-inflammatory markers including TNF-α and TGF-β; and (iii) the expression of miR-200b and miR-155. Consistently, the pharmacological inhibition of EZH2 by 3-Deazaneplanocin A (DZNep) significantly reduces EZH2 expression/activity, while it increases lipid accumulation, inflammatory molecules and microRNAs. In conclusion, the results of this study suggest that the defective activity of EZH2 can enhance the NAFLD development by favouring steatosis and the de-repression of the inflammatory genes and that of specific microRNAs.

Differential toxicity, conformation and morphology of typical initial aggregation states of Aβ1-42 and Aβpy3-42 beta-amyloids.

Among the different species of water-soluble β-peptides (Aβ1-42, Aβ1-40 and N-terminal truncated Aβ-peptides), Aβpy3-42 is thought to play a relevant role in Alzheimers pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aβpy3-42, we separated different aggregation states of Aβ1-42 and Aβpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates. Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical β-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given β-peptide and among the aggregate of the two β-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aβ1-42 the most toxic aggregate is la, while in the case of Aβpy3-42 both sa and la are equally toxic. Aβ aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aβpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aβpy3-42 peptide.

Probing cytoskeleton organisation of neuroblastoma cells with single-cell force spectroscopy.

Single‐cell force spectroscopy is an emerging technique in the field of biomedicine because it has proved to be a unique tool to obtain mechanical and functional information on living cells, with force resolution up to single molecular bonds. This technique was applied to the study of the cytoskeleton organisation of neuroblastoma cells, a life‐threatening cancer typically developing during childhood, and the results were interpreted on the basis of reference experiments on human embryonic kidney cell line. An intimate connection emerges among cellular state, cytoskeleton organisation and experimental outcome that can be potentially exploited towards a new method for cancer stadiation of neuroblastoma cells. Copyright

Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma

Background:Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours.Methods:Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines.Results:Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments.Conclusion:Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

Global profiling of viral and cellular non-coding RNAs in Epstein-Barr virus-induced lymphoblastoid cell lines and released exosome cargos.

The human EBV-transformed lymphoblastoid cell line (LCL), obtained by infecting peripheral blood monocular cells with Epstein-Barr Virus, has been extensively used for human genetic, pharmacogenomic, and immunologic studies. Recently, the role of exosomes has also been indicated as crucial in the crosstalk between EBV and the host microenvironment. Because the role that the LCL and LCL exosomal cargo might play in maintaining persistent infection, and since little is known regarding the non-coding RNAs of LCL, the aim of our work was the comprehensive characterization of this class of RNA, cellular and viral miRNAs, and cellular lncRNAs, in LCL compared with PBMC derived from the same donors. In this study, we have demonstrated, for the first time, that all the viral miRNAs expressed by LCL are also packaged in the exosomes, and we found that two miRNAs, ebv-miR-BART3 and ebv-miR-BHRF1-1, are more abundant in the exosomes, suggesting a microvescicular viral microRNA transfer. In addition, lncRNA profiling revealed that LCLs were enriched in lncRNA H19 and H19 antisense, and released these through exosomes, suggesting a leading role in the regulation of the tumor microenvironment.

Targeting ABCB1 and ABCC1 with their Specific Inhibitor CBT-1® can Overcome Drug Resistance in Osteosarcoma.

Clinical treatment response achievable with conventional chemotherapy in high-grade osteosarcoma (OS) is severely limited by the presence of intrinsic or acquired drug resistance, which in previous studies has been mainly addressed for overexpression of ABCB1 (MDR1/P-glycoprotein). This study was aimed to estimate the impact on OS drug resistance of a group of ATP binding cassette (ABC) transporters, which in other human tumors have been associated with unresponsiveness to the drugs that represent the backbone of multidrug treatment regimens for OS (doxorubicin, methotrexate, cisplatin). By using a group of 6 drug-sensitive and 20 drug-resistant human OS cell lines, the most relevant transporter which proved to be associated with the degree of drug resistance in OS cells, in addition to ABCB1, was ABCC1. We therefore evaluated the in vitro activity of the orally administrable ABCB1/ABCC1 inhibitor CBT-1(®) (Tetrandrine, NSC-77037). We found that in our OS cell lines this agent was able to revert the ABCB1/ABCC1-mediated resistance against doxorubicin, as well as against the drugs used in second-line OS treatments that are substrates of these transporters (taxotere, etoposide, vinorelbine). Our findings indicated that inhibiting ABCB1 and ABCC1 with CBT-1(®), used in association with conventional chemotherapeutic drugs, may become an interesting new therapeutic option for unresponsive or relapsed OS patients.