DNMT3B in vitro knocking-down is able to reverse embryonal rhabdomyosarcoma cell phenotype through inhibition of proliferation and induction of myogenic differentiation

Aberrant DNA methylation has been frequently observed in many human cancers, including rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. To date, the expression and function of the de novo DNA methyltransferase (DNMT) 3B in RMS have not yet been investigated. Our study show for the first time a significant up-regulation of DNMT3B levels in 14 RMS tumour samples and 4 RMS cell lines in comparison to normal skeletal muscle. Transfection of RD and TE671 cells, two in vitro models of embryonal RMS (ERMS), with a synthetic DNMT3B siRNA decreased cell proliferation by arresting cell cycle at G1 phase, as demonstrated by the reduced expression of Cyclin B1, Cyclin D1 and Cyclin E2, and by the concomitant up-regulation of the checkpoint regulators p21 and p27. DNMT3B depletion also impaired RB phosphorylation status and decreased migratory capacity and clonogenic potential. Interestingly, DNMT3B knock-down was able to commit ERMS cells towards myogenic terminal differentiation, as confirmed by the acquisition of a myogenic-like phenotype and by the increased expression of the myogenic markers MYOD1, Myogenin and MyHC. Finally, inhibition of MEK/ERK signalling by U0126 resulted in a reduction of DNMT3B protein, giving evidence that DNMT3B is a down-stream molecule of this oncogenic pathway.Taken together, our data indicate that altered expression of DNMT3B plays a key role in ERMS development since its silencing is able to reverse cell cancer phenotype by rescuing myogenic program. Epigenetic therapy, by targeting the DNA methylation machinery, may represent a novel therapeutic strategy against RMS

Crizotinib-induced antitumour activity in human alveolar rhabdomyosarcoma cells is not solely dependent on ALK and MET inhibition

BACKGROUND: Rhabdomyosarcoma (RMS) is the most commonly diagnosed malignant soft tissue tumour in children and adolescents. Aberrant expression of Anaplastic Lymphoma Kinase (ALK) and MET gene has been implicated in the malignant progression of RMS, especially in the alveolar subtype. This observation suggests that crizotinib (PF-02341066), a kinase inhibitor against ALK and MET, may have a therapeutic role in RMS, although its antitumour activity in this malignancy has not yet been studied. METHODS: RH4 and RH30 alveolar RMS (ARMS) cell lines were treated with crizotinib and then assessed by using proliferation, viability, migration and colony formation assays. Multiple approaches, including flow cytometry, immunofluorescence, western blotting and siRNA-based knock-down, were used in order to investigate possible molecular mechanisms linked to crizotinib activity. RESULTS: In vitro treatment with crizotinib inhibited ALK and MET proteins, as well as Insulin-like Growth Factor 1 Receptor (IGF1R), with a concomitant robust dephosphorylation of AKT and ERK, two downstream kinases involved in RMS cell proliferation and survival. Exposure to crizotinib impaired cell growth, and accumulation at G2/M phase was attributed to an altered expression and activation of checkpoint regulators, such as Cyclin B1 and Cdc2. Crizotinib was able to induce apoptosis and autophagy in a dose-dependent manner, as shown by caspase-3 activation/PARP proteolytic cleavage down-regulation and by LC3 activation/p62 down-regulation, respectively. The accumulation of reactive oxygen species (ROS) seemed to contribute to crizotinib effects in RH4 and RH30 cells. Moreover, crizotinib-treated RH4 and RH30 cells exhibited a decreased migratory/invasive capacity and clonogenic potential. CONCLUSIONS: These results provide a further insight into the molecular mechanisms affected by crizotinib in ARMS cells inferring that it could be a useful therapeutic tool in ARMS cancer treatment

Quantitative Real-Time PCR detection of TRPV1–4 gene expression in human leukocytes from healthy and hyposensitive subjects

<p>Abstract</p> <p>Background</p> <p>Besides functioning as chemosensors for a broad range of endogenous and synthetic ligands, transient receptor potential vanilloid (TRPV) 1–4 channels have also been related to capsaicin (TRPV1), pain, and thermal stimuli perception, and itching sensation (TRPV1–4). While the expression of the TRPV1–4 genes has been adequately proved in skin, sensory fibres and keratinocytes, less is known about TRPV3 and TRPV4 expression in human blood cells.</p> <p>Results</p> <p>To study the gene expression of TRPV1–4 genes in human leukocytes, a quantitative Real-Time PCR (qRT-PCR) method, based on the calculation of their relative expression, has been developed and validated. The four commonly used house-keeping genes (HKGs), β-Actin (Act-B), glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxanthine ribosyltransferase (HPRT1), and cyclophilin B (hCyPB), were tested for the stability of their expression in several human leukocyte samples, and used in the normalization procedure to determine the mRNA levels of the TRPV 1–4 genes in 30 healthy subjects. cDNAs belonging to all the TRPV1–4 genes were detected in leukocytes but the genes appear to be expressed at different levels. Our analysis did not show significant sex differences in TRPV1–4 cDNA levels in the 30 healthy subjects. The same qRT-PCR assay was used to compare TRPV1–4 expression between healthy controls and patients hyposensitive to capsaicin, pain and thermal stimuli: an almost doubled up-regulation of the TRPV1 gene was found in the pathological subjects.</p> <p>Conclusion</p> <p>The qRT-PCR assay developed and tested in this study allowed us to determine the relative expression of TRPV1–4 genes in human leukocytes: TRPV3 is the least expressed gene of this pool, followed by TRPV4, TRPV1 and TRPV2. The comparison of TRPV1–4 gene expression between two groups of healthy and hyposensitive subjects highlighted the evident up-regulation of TRPV1, which was almost doubly expressed (1.9× normalized fold induction) in the latter group. All the four house-keeping genes tested in this work (Act-B, GAPDH, hCyPB, HPRT1) were classified as optimal controls and showed a constant expression in human leukocytes samples. We recommend the use of these genes in similar qRT-PCR studies on human blood cells.</p

LPS-induced TNF-α factor mediates pro-inflammatory and pro-fibrogenic pattern in non-alcoholic fatty liver disease

Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH).We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1β, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1β transcription exclusively required LITAF expression/activity. Finally, IL-1β levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH.In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1β levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH

MO030IDENTIFICATION OF REGULATED MRNAS AND MIRNAS IN GLOMERULI ISOLATED FROM AN FSGS-LIKE ZEBRAFISH MODEL

Abstract Background and Aims The zebrafish (Danio rerio) is a powerful animal model to study glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to become transparent, in vivo observation of these animals is possible. At 48 hours post fertilization, zebrafish larvae develop a single glomerulus which is attached to a pair of tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane and interdigitating podocyte foot processes bridged by a slit diaphragm. By using genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease like focal segmental glomerulosclerosis (FSGS) directly in vivo. FSGS is characterized by podocyte loss, the effacement of their foot processes as well as scarring of the glomerulus. To study FSGS in zebrafish larvae, we induced podocyte detachment by the use of a zebrafish strain expressing the enzyme nitroreductase converting metronidazole into a toxic substance specifically in podocytes. The aim of our study was to collect glomeruli for the identification of mRNAs as well as miRNAs by RNA_Seq that are up- and down-regulated in the glomeruli of this FSGS-like disease model. Method The transgenic zebrafish strain Cherry (Tg(nphs2:GAL4); Tg(UAS:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was utilized. After addition of metronidazole (MTZ) into the tank water, MTZ is converted into a cytotoxin by NTR leading to dose-dependent apoptosis exclusively in podocytes. Cherry larvae were treated at 4 days post fertilization (dpf) for 48 h with 80 µM MTZ. MTZ-treated and control larvae were homogenized at 6 dpf. The cell suspension was diluted, and red-fluorescent glomeruli were collected using a micropipette and a microscope. Total RNA was isolated, and integrity was checked by a Bioanalyzer. Libraries were generated with a MACE kit and True Quant small RNA seq kit by GenXPro. Constructs were amplified by PCR and sequenced on an Illumina Hiseq 2000. Normalization and statistical analysis for differential gene expression were done using DESeq2. Results Zebrafish larvae showed severe whole-body edema, proteinuria, loss of podocytes and an increased mortality rate after MTZ-treatment. The glomerular histology resembled mammalian FSGS. We found that only the RNA of manually collected glomeruli had an excellent quality. Using RNA_Seq, we identified a total of 16941 genes. DESeq2 analysis showed 494 up-regulated and 473 down-regulated genes. Gene ontology (GO) enrichment analysis of up-regulated genes revealed a total of 167 that are significantly enriched in GO terms (e.g. metabolic processes, immune response and ion transport). Down-regulated genes were enriched in 14 GO terms and most of them are linked to normal glomerular function and the slit diaphragm. DESeq2 analysis identified 200 miRNAs of 777 small RNAs. Some of these miRNA are already described to be regulated in different glomerular diseases like FSGS, lupus nephritis, IgA nephropathy and diabetic nephropathy. Conclusion We analyzed isolated glomeruli from transgenic zebrafish larvae that developed a FSGS-like disease. By sequencing, we have found mRNAs and miRNAs that were significantly regulated after the onset of disease. Detailed knowledge of these mRNAs and miRNA-based gene regulation will help to uncover the pathomechanism as well as to develop therapeutics for the treatment of FSGS

Role of the endothelin-1 system in the luteolytic process of pseudopregnant rabbits

The aim of this study was to better understand the role of the endothelin-1 (ET-1) system in the process of controlling the corpora lutea (CL) life span in rabbits. ET-1 (10 microg iv) administration at d 9 and 12 of pseudopregnancy induced a functional luteolysis within 24 h of injection, but it was ineffective at both d 4 and 6. Pretreatments with Bosentan, a dual ET(A)/ET(B) receptor antagonist, or cyclooxygenase (COX) inhibitor blocked the luteolytic action of ET-1 but not that induced by prostaglandin F2alpha (PGF2alpha). In CL cultured in vitro, ET-1 increased (P </= 0.01) both PGF(2alpha) production and luteal nitric oxide synthase activity but decreased (P < or = 0.01) progesterone release. Addition of ET(A) receptor antagonist BQ123 or COX inhibitor blocked the ET-1 luteolytic effects. Positive staining for ET-1 receptors was localized in ovarian blood vessels, granulosa cells of large follicles, and luteal cells. Immunoblot analysis of ET-1 receptor protein revealed a strong band of approximately 48 kDa in d-9 CL. Up to d 6 of pseudopregnancy, ET-1 mRNA abundance in CL was poorly expressed but then increased (P < or = 0.01) at d 9 and 13. ET(A)-receptor transcript increased (P < or = 0.01) at d 6, remained at the same level up to d 13, and then declined to the lowest (P < or = 0.01) levels at d 22. ET(B)-receptor mRNA increased (P < or = 0.01) throughout the late-luteal stage from d 13 up to d 18. Our data suggest that the luteolytic action of ET-1 may be a result of PGF2alpha synthesis from both luteal and accessory cells, via the COX pathways

How skill expertise shapes the brain functional architecture: an fMRI study of visuo-spatial and motor processing in professional racing-car and naïve drivers

The present study was designed to investigate the brain functional architecture that subserves visuo-spatial and motor processing in highly skilled individuals. By using functional magnetic resonance imaging (fMRI), we measured brain activity while eleven Formula racing-car drivers and eleven ‘naïve’ volunteers performed a motor reaction and a visuo-spatial task. Tasks were set at a relatively low level of difficulty such to ensure a similar performance in the two groups and thus avoid any potential confounding effects on brain activity due to discrepancies in task execution. The brain functional organization was analyzed in terms of regional brain response, inter-regional interactions and blood oxygen level dependent (BOLD) signal variability. While performance levels were equal in the two groups, as compared to naïve drivers, professional drivers showed a smaller volume recruitment of task-related regions, stronger connections among task-related areas, and an increased information integration as reflected by a higher signal temporal variability. In conclusion, our results demonstrate that, as compared to naïve subjects, the brain functional architecture sustaining visuo-motor processing in professional racing-car drivers, trained to perform at the highest levels under extremely demanding conditions, undergoes both ‘quantitative’ and ‘qualitative’ modifications that are evident even when the brain is engaged in relatively simple, non-demanding tasks. These results provide novel evidence in favor of an increased ‘neural efficiency’ in the brain of highly skilled individuals

Association between Serum Atypical Fibroblast Growth Factors 21 and 19 and Pediatric Nonalcoholic Fatty Liver Disease

Atypical fibroblast growth factors (FGF) 21 and 19 play a central role in energy metabolism through the mediation of Klotho coreceptor. Contradictory findings are available about the association of FGF21 and FGF19 with nonalcoholic fatty liver disease (NAFLD) in humans. We investigated the association of serum FGF21, FGF19 and liver Klotho coreceptor with non-alcoholic steatohepatitis (NASH) and fibrosis in children with NAFLD. Serum FGF21 and FGF19 were measured in 84 children with biopsy-proven NAFLD and 23 controls (CTRL). The hepatic expression of Klotho coreceptor was measured in 7 CTRL, 9 patients with NASH (NASH+) and 11 patients without NASH (NASH-). FGF21 and FGF19 showed a tendency to decrease from CTRL (median FGF21 = 196 pg/mL; median FGF19 = 201 pg/mL) to NASH- (FGF21 = 89 pg/mL; FGF19 = 81 pg/mL) to NASH+ patients (FGF21 = 54 pg/mL; FGF19 = 41 pg/mL) (p&lt;0.001 for all comparisons) and were inversely associated with the probability of NASH and fibrosis in children with NAFLD. The hepatic expression of Klotho coreceptor was inversely associated with NASH (R2 = 0.87, p&lt;0.0001) and directly associated with serum FGF21 (R2 = 0.57, p&lt;0.0001) and FGF19 (R2 = 0.67, p&lt;0.0001). In conclusion, serum FGF19 and FGF21 and hepatic Klotho expression are inversely associated with hepatic damage in children with NAFLD and these findings may have important implications for understanding the mechanisms of NAFLD progression. © 2013 Alisi et al