Long Noncoding RNA GAS5: A Novel Marker Involved in Glucocorticoid Response

Glucocorticoids (GCs) exert their effects through regulation of gene expression after activation in the cytoplasm of the glucocorticoid receptor (GR) encoded by NR3C1 gene. A negative feedback mechanism resulting in GR autoregulation has been demonstrated through the binding of the activated receptor to intragenic sequences called GRE-like elements, contained in GR gene. The long noncoding RNA growth arrest-specific transcript 5 (GAS5) interacts with the activated GR suppressing its transcriptional activity. The aim of this study was to evaluate the possible role of GAS5 and NR3C1 gene expression in the antiproliferative effect of methylprednisolone in peripheral blood mononuclear cells and to correlate the expression with individual sensitivity to GCs. Subjects being poor responders to GCs presented higher levels of GAS5 and NR3C1 in comparison with good responders. We suggest that abnormal levels of GAS5 may alter GC effectiveness, probably interfering with the mechanism of GR autoregulation

Carbamazepine-induced thrombocytopenic purpura in a child: Insights from a genomic analysis

To the Editor, Carbamazepine is an effective anticonvulsant and has a relatively low incidence of adverse effects, although it occasionally causes hema- tologic disorders. We herein describe a patient with carbamazepine- induced thrombocytopenic purpura that was investigated by pharma- cological, immunological and genomic assays

Nanostrutture di carbonio come vettori per farmaci antitumorali

2011/2012Carbon nanotechnology has evolved into a truly interdisciplinary field, bridging material science with medicine. Fullerenes (C60) play a major role in this field and are currently explored for biomedical applications such as radiopharmaceuticals and contrast agents, gene delivery and as carriers for chemotherapeutics. Five fullerene derivatives (F1, F2, F3, F4 and F5), functionalized by 1,3-dipolar cycloaddition of azomethine ylides to the C60 cage, were studied in vitro for their toxicity in a number of cell types and with different assays. Cell cytotoxicity on human mammary carcinoma cell line (MCF7), evaluated with the MTT and NRU tests and further confirmed by a flow cytometry approach with DiOC6 and PI probes, showed that derivative F2 was free of necrotic or apoptotic effects even after a long lasting cell exposure. F3 (differing from F2 for an additional positive charge obtained by quaternarization of the pyrrolidinic nitrogen by introducing a methyl group) was more toxic compared to the other compounds in all cellular models employed (HCT116, MCF7, MCF7/ADR, HT-29, H460, B16F10 and MDAMB231). Its IC50 is 20 µM after 72 hr of incubation by MTT test, and cell accumulation in the G1 phase and arrest in G0 phase (30%) was also observed. The mechanism of cellular uptake (studied with a fluorescein-bearing derivative of F2, hereafter called derivative F2-FITC), and the intracellular distribution were analyzed on MCF7 cell line. The studies of F2 biological effects have shown that this compound is able to enter the treated cells, probably by passive diffusion, to distribute within the cell cytoplasm, without getting into the nucleoplasm or into organelles such as lysosomes and mitochondria; these processes were evaluated by flow cytometry and confirmed by confocal microscopy. Experiments were performed on a multidrug resistant human mammary carcinoma cell line MCF7/ADR, a sub-line resistant to Doxorubicin because of the overexpression of the P-glycoprotein (P-gp) extrusion pump. The choice of these cells was based on our objective to conjugate the anticancer drug Doxorubicin to the fullerene vector in order to overcome adriamycin resistance. The F2 cellular uptake on MCF7/ADR and its maintenance of a constant concentration into cells seem to be insensitive to the presence of P-gp over-expression. These data suggest the possibility for derivative F2 to be used as carrier for anticancer drugs. The experiments on conjugated F2-DOX were made above all to verify if the activity of the drug linked to the fullerene remained the same as the free drug. Furthermore we studied the cellular uptake of F2-DOX in both MCF7 and MCF/ADR lines. Cytotoxicity tests have shown that F2-DOX has an irrelevant activity compared to free drug because Doxorubicin cannot get into the nucleus to perform its activity as it remains linked to F2. Nevertheless, the internalization of F2-DOX is higher in MCF7/ADR compared to the free drug. Fluorescent microscopy technique suggested that the F2-DOX inactivity might be associated with the stability of the bond between the carrier and the drug, which is not released and so is localized in the cytosol, as we have also observed for F2. Since many studies showed contradictory results and the molecular and cellular mechanisms of the cytotoxicity of this class of nanomaterials are not yet fully understood we performed a whole genome expression analysis, by high throughput RNA sequencing, using Illumina technologies. All together, the RNA-seq expression data confirmed the experimental evidence collected with previous in vitro studies showing that F3 is definitely the derivative causing more alterations on MCF7 cells on both the molecular and the cellular levels. However, also F2 is capable of affecting the same molecular pathways, although to a much lower intensity, since neither cytotoxic effects nor cell cycle arrest could be documented. The most important, and somewhat unexpected, result of our analysis was certainly the lack of molecular evidence concerning the activation of the main cellular pathways leading to cell death and often linked to fullerene toxicity in literature. In fact apoptosis, autophagy and ROS damage does not seem to be included among the most relevant molecular effects of F3, which on the contrary are mainly linked to the arrest of the transcription and protein synthesis machineries, leading to the entry in the G0 cell cycle phase. The RNA-seq analysis was able to identify several additional effects of fullerenes which had not been previously described, offering a complete overview of the gene expression alterations induced by these compounds on a whole-transcriptome level. Therefore the combination of large scale molecular analysis and the main viability assays might represent a valuable tool for a better understanding of the toxicity of fullerenes and other nanomaterials.L’impiego di nanostrutture di carbonio, come i fullereni, in campo biomedico è di forte interesse, sia per le sue possibili applicazioni terapeutiche che diagnostiche. Un potenziale vantaggio di questi composti è la possibilità di essere strutturati per essere facilmente internalizzati dalle cellule e grazie alla loro ampia area superficiale e volume interno possono agire come sistemi di drug delivery per il trasporto di farmaci. Cinque fullereni funzionalizzati (F1, F2, F3, F4 e F5) sono stati studiati in vitro per la loro tossicità in un certo numero di linee cellulari e con dosaggi e tempi di incubazione diversi con l’obiettivo di valutare se gli effetti tossici o meno di questi composti possano variare a seconda dalla linea cellulare utilizzata. I modelli di studio utilizzati sono linee cellulari neoplastiche di carcinoma mammario umano (MCF7, MCF7/ADR e MDAMB231), di adenocarcinoma umano di colon (HT-29 e HCT116), di adenocarcinoma polmonare umano non a piccole cellule (H460) e di melanoma murino (B16F10). Gli studi di citotossicità hanno permesso di individuare un fullerene che presenta una limitata tossicità (F2) con il quale sono state eseguite prove citofluorimetriche sulla linea MCF7 che hanno dimostrato come sia in grado di attraversare le membrane plasmatiche e di accumularsi nelle cellule già dopo un’ora, aumentando in quantità fino a 12 ore. L’analisi è stata condotta fino alle 72 ore, tempo in cui il valore di fluorescenza del fullerene sembra rimanere stabile. Mediante microscopia a fluorescenza, sono stati condotti studi di distribuzione sub-cellulare, dai quali è emerso che F2 non colocalizza né a livello mitocondriale nè lisosomiale; quest’ultima evidenza lo renderebbe un vettore stabile per farmaci acido-labili. Inoltre, la mancata internalizzazione del fullerene a livello nucleare escluderebbe la possibilità di un danno diretto a carico del materiale genetico. In parallelo sono stati condotti i primi esperimenti sul coniugato F2-DOX ma le prove di citotossicità hanno indicato che il coniugato, nella linea MCF7 ed MCF7/ADR, ha attività irrilevante rispetto alla Doxorubicina libera. Tuttavia risulta che la Doxorubicina trasportata da F2 viene internalizzata maggiormente nella linea MCF7/ADR rispetto alla linea MCF7, contrariamente a quanto avviene con il farmaco libero. È possibile quindi affermare che F2 si potrebbe dimostrare un valido vettore per eludere il problema della multidrug resistence nei confronti di farmaci antitumorali causata dall’attività estrusiva della pompa P-gp. L’ostacolo da superare rimane quindi la scelta del farmaco da coniugare visto che questo ipotetico vettore non entra nel nucleo (e che quindi la Doxorubicina e altri antitumorali ad azione nucleare non potrebbero svolgere le proprie funzioni) nelle linee studiate. In alternativa si potrebbe pensare di legare il farmaco al fullerene attraverso un legame più debole in modo da facilitarne il rilascio citosolico. Dal momento che molti studi di tossicità hanno mostrato risultati contraddittori ed i meccanismi molecolari di questa classe di nanomateriali non sono ancora pienamente compresi abbiamo effettuato un’analisi dell’intero trascrittoma sfruttando la tecnologia Illumina. I dati ottenuti mediante l’utilizzo di questa tecnica di next generation sequencing (RNA-Seq) hanno confermato l'evidenza sperimentale raccolta con i precedenti studi in vitro dimostrando che F3 è sicuramente il derivato che causa più alterazioni sulle MCF7, sia a livello molecolare che cellulare. Tuttavia è stato osservato che F2 è in grado di pregiudicare le stesse vie molecolari, anche se con un'intensità molto più bassa. Il risultato più importante, e inaspettato, emerso dalle nostre analisi è stato certamente la mancanza di prove molecolari riguardanti l'attivazione delle vie principali che portano alla morte cellulare e spesso legati alla tossicità fullerene, come descritto in letteratura. Apoptosi, autofagia o stress da produzione di ROS non sembrano essere inclusi tra gli effetti molecolari più rilevanti di F3, che al contrario sono principalmente legati all'arresto della trascrizione e alla sintesi proteica, fenomeni che conducono all’arresto della divisione e all'entrata in fase G0. L'analisi di RNA-seq è stata in grado di identificare diversi effetti addizionali dei fullereni che non erano state precedentemente descritti, offrendo una panoramica completa delle alterazioni di espressione genica indotte da questi composti sull’intero trascrittoma. Quindi la combinazione di analisi molecolari su larga scala e saggi di vitalità potrebbe rappresentare un valido strumento per una migliore comprensione della tossicità dei fullereni ed altri nanomateriali.XXV Ciclo198

RNA-seq analysis of the whole transcriptome of MDA-MB-231 mammary carcinoma cells exposed to the antimetastatic drug NAMI-A

Gene expression profiling has been introduced into drug development to understand the activity of chemical entities in pre-clinical settings. The present study investigates the changes induced in gene expression by the ruthenium-based compound NAMI-A. The genes differentially expressed by NAMI-A are evaluated through whole-transcriptome analysis and RNA-sequencing in the metastatic MDA-MB-231 mammary carcinoma cells, in comparison to the non-tumorigenic HBL-100 mammary gland cells. NAMI-A treatment rapidly induces a relevant gene up-regulation that quickly returns to normal values. These changes differ between MDA-MB-231 and HBL-100 cells, highlighting the selectivity of the NAMI-A induced transcriptional perturbation in the invasive rather than in the non-tumorigenic cells. The transcriptional response, in the invasive MDA-MB-231 cells, comprises a set of early-response transcription factors and reveals a pharmacological signature in good agreement with the most peculiar NAMI-A behavior as a metastasis inhibitor such as cell cycle regulation and ECM remodeling. Globally, the results of this study indicate some transcription factors influencing the expression and activity of many downstream genes and proteins fundamentally involved in the functional effects of NAMI-A in vitro and in vivo

Clinical Application of Thiopurine Pharmacogenomics in Pediatrics

Background: Thiopurine drugs are used for the treatment of pediatric diseases. Inter-individual differences in the metabolism of these drugs greatly influence the risk of thiopurine induced toxicity and therapy failure. These differences are the consequence of genomic, epigenomic and transcriptomic variability among patients. Pharmacogenomics aims to individualize therapy according to the specific genetic signature of a patient. Treatment protocols based on thiopurine drugs have already been improved by applying pharmacogenomics in pediatric clinical practice. Objective: The aim of this review was to summarize the application of thiopurine pharmacogenomics in pediatric patients suffering from acute leukemias, different types of autoimmune and inflammatory diseases, as well as in posttransplant care. Methods: We searched PubMed/Medline database to identify thiopurine pharmacogenomic markers clinically relevant in pediatric diseases. Results: TPMT and NUDT15 pharmacogenomic testing is done in pediatric care, contributing to the reduction of thiopurine induced toxicity. Data on numerous novel potential pharmacogenomic markers relevant for optimization of thiopurine treatment are still controversial (ITPA, ABCC4, NT5C2, PRPS1, GSTM1, FTO gene variants). Majority of evidences regarding thiopurine pharmacogenomics in pediatrics have been acquired by studying acute lymphoblastic leukemia and inflammatory bowel disease. For other pediatric diseases, namely acute myeloid leukemia, non-Hodgkin lymphoma, juvenile idiopathic arthritis, atopic dermatitis, juvenile autoimmune hepatitis and renal allograft transplantation, data are still scarce. Conclusion: Thiopurine pharmacogenomics has shown to be one of the best examples of successful application of pharmacogenomics in pediatrics

Pharmacotranscriptomic Biomarkers in Glucocorticoid Treatment of Pediatric Inflammatory Bowel Disease

9siPharmacotranscriptomics aims to reach more accurate drug dosing based on interindividual transcriptome variations. Here, we provide an overview of RNA biomarkers that could predict the response to glucocorticoids (GCs), considered the standard for treatment of inflammatory bowel diseases (IBD), both in adult and pediatric patients. Although new biological agents are very effective in IBD treatment, GCs are still widely used for induction of remission in patients with moderate to severe disease. It is important to identify patients that are poor responders to GCs therapy, because suboptimal response is frequent and associated with various side effects. A number of genetic variants related to GC mechanism of action has been studied. However, the majority of reported associations are not consistent. In this regard, pharmacogenomic research is now exploring the world of RNAs. An appropriate regulation of the transcriptome, which mainly comprises mRNAs and non-coding RNAs that control gene expression, has a strong impact in the modulation of GC activity.reservedmixedLucafò, Marianna; Stankovic, Biljana; Kotur, Nikola; Di Silvestre, Alessia; Martelossi, Stefano; Ventura, Alessandro; Zukic, Branka; Pavlovic, Sonja; Decorti, GiulianaLucafò, Marianna; Stankovic, Biljana; Kotur, Nikola; Di Silvestre, Alessia; Martelossi, Stefano; Ventura, Alessandro; Zukic, Branka; Pavlovic, Sonja; Decorti, Giulian

Causes of Treatment Failure in Children With Inflammatory Bowel Disease Treated With Infliximab: A Pharmacokinetic Study

17noObjectives: Anti-tumor necrosis factor antibodies have led to a revolution in the treatment of inflammatory bowel diseases (IBD); however, a sizable proportion of patients does not respond to therapy. There is increasing evidence suggesting that treatment failure may be classified as mechanistic (pharmacodynamic), pharmacokinetic, or immune-mediated. Data regarding the contribution of these factors in children with IBD treated with infliximab (IFX) are still incomplete. The aim was to assess the causes of treatment failure in a prospective cohort of pediatric patients treated with IFX. Methods: This observational study considered 49 pediatric (median age 14.4) IBD patients (34 Crohn disease, 15 ulcerative colitis) treated with IFX. Serum samples were collected at 6, 14, 22 and 54 weeks, before IFX infusions. IFX and anti-infliximab antibodies (AIA) were measured using enzyme linked immunosorbent assays. Disease activity was determined by Pediatric Crohn's Disease Activity Index or Pediatric Ulcerative Colitis Activity Index. Results: Clinical remission, defined as a clinical score 3.11 μg/mL emerged as the strongest predictor of sustained clinical remission. AIA concentrations were correlated inversely with IFX concentrations and directly with adverse reactions. Conclusions: Most cases of therapeutic failure were associated with low serum drug levels. IFX trough levels at the end of induction are associated with sustained long-term response.partially_openopenNaviglio, Samuele; Lacorte, Doriana; Lucafò, Marianna; Cifù, Adriana; Favretto, Diego; Cuzzoni, Eva; Silvestri, Tania; Pozzi Mucelli, Martina; Radillo, Oriano; Decorti, Giuliana; Fabris, Martina; Bramuzzo, Matteo; Taddio, Andrea; Stocco, Gabriele; Alvisi, Patrizia; Ventura, Alessandro; Martelossi, StefanoNaviglio, Samuele; Lacorte, Doriana; Lucafò, Marianna; Cifù, Adriana; Favretto, Diego; Cuzzoni, Eva; Silvestri, Tania; Pozzi Mucelli, Martina; Radillo, Oriano; Decorti, Giuliana; Fabris, Martina; Bramuzzo, Matteo; Taddio, Andrea; Stocco, Gabriele; Alvisi, Patrizia; Ventura, Alessandro; Martelossi, Stefan

Imidazo[2,1-b]benzothiazol derivatives as potential allosteric inhibitors of the glucocorticoid receptor

Glucocorticoid receptor (GCR) transactivation reporter gene assays were used as an initial high-throughput screening on a diversified library of 1200 compounds for their evaluation as GCR antagonists. A class of imidazo[2,1-b]benzothiazole and imidazo[2,1-b]benzoimidazole derivatives were identified for their ability to modulate GCR transactivation and anti-inflammatory transrepression effects utilizing GCR and NF-\u3baB specific reporter gene assays. Modeling studies on the crystallographic structure of the GCR ligand binding domain provided three new analogues bearing the tetrahydroimidazo[2,1-b]benzothiazole scaffold able to antagonize the GCR in the presence of dexamethasone (DEX) and also defined their putative binding into the GCR structure. Both mRNA level measures of GCR itself and its target gene GILZ, on cells treated with the new analogues, showed a GCR transactivation inhibition, thus suggesting a potential allosteric inhibition of the GCR