Profilo farmacogenetico di determinanti molecolari dell'attivita di cisplatino e gemcitabina in pazienti affetti da tumore polmonare non a piccole cellule

Il carcinoma polmonare rappresenta un problema socio-sanitario di grande rilievo, essendo la prima causa di morte per neoplasia. Ancor oggi la diagnosi di tale tumore è sfortunatamente tardiva con il 75% dei pazienti che si presenta in stadio localmente avanzato o metastatico. Il carcinoma polmonare non a piccole cellule ( non small cell lung cancer, NSCLC) rappresenta la variante istologica più frequente (80% dei casi di tumore polmonare). Per questi pazienti il trattamento chemioterapico determina un prolungamento della sopravvivenza e un miglioramento della qualità della vita rispetto alla sola terapia di supporto, identificandosi come standard terapeutico. L’ individuazione del migliore trattamento chemioterapico per questo subset di pazienti rappresenta pertanto una delle principali sfide della ricerca oncologica. I regimi polichemioterapici si possono dividere schematicamente in tre generazioni in relazione all’ introduzione nel corso degli anni di nuovi agenti chemioterapici. Con l’avvento dei regimi di terza generazione, il trattamento del NSCLC avanzato sembra aver raggiunto un plateau, mancando infatti chiare dimostrazioni di superiorità di un regime di ultima generazione rispetto ad un altro. Tra questi l’associazione cisplatino e gemcitabina rappresenta uno dei regimi standard più utilizzati in considerazione del suo favorevole rapporto costo-beneficio. Al fine di migliorare i risultati del trattamento chemioterapico in termini di attività ed efficacia, una possibilità consiste nell’individuazione di parametri predittivi che ci consentano di identificare il miglior trattamento per il singolo paziente. Tra i vari parametri predittivi valutabili, un crescente interesse è stato rivolto a quelli di carattere genetico, anche grazie all’avvento di nuove tecniche di biologia molecolare e al sequenziamento del genoma umano che ha dato nuovo impulso a studi di farmacogenetica e farmacogenomica. Nell’ambito di tali studi, si collocano le analisi dei polimorfismi del singolo nucleotide (SNPs) a livello di regioni codificanti del genoma, che possono influenzare l’espressione e la funzione delle proteine. Si parla di polimorfismo genico quando esistono, nella popolazione normale, geni mutati o variati con una frequenza superiore all’1%. Sulla base di queste considerazioni, in questa tesi sono stati effettuati 2 studi: 1) un’analisi di SNPs nei geni codificanti per la subunità 1 della ribonucleotide redattasi (RRM1) e la citidina deaminasi CDA, che sono rispettivamente il principale enzima del catabolismo e il target della gemcitabina; e 2) uno studio valutare l’espressione di determinanti molecolari coinvolti nel meccanismo di azione di gemcitabina e cisplatino in pazienti affetti dai due tipi istologici principali di NSCLC, adenocarcinomi e carcinomi squamocellulari. In particolare, pazienti affetti da NSCLC trattati con regime chemioterapici con gemcitabina e cisplatino o gemcitabina e carboplatino sono stati genotipizzati per gli alleli degli SNPs CDA A79C e RRM1 G2464A mediante Real Time PCR con sonde Taqman. Lo scopo di questo studio è stato quello di correlare il genotipo con la risposta clinica alla chemioterapia. La valutazione degli SNPs in una iniziale casistica di 65 pazienti ha evidenziato una percentuale di risposte cliniche e una sopravvivenza significativamente superiore nei pazienti con genotipo CDA A79A. Tali risultati non sono stati però confermate dalle analisi effettuate in una più ampia casistica di 226 pazienti in trattamento presso l’Ospedale di Torino. Lo studio dei livelli di espressione genica è stata invece effettuata in tessuti di 70 pazienti affetti da NSCLC arruolati presso l’Istituto Europeo di Oncologia di Milano. In particolare, mediante Real Time PCR è stata valutata l’espressione genica di ERCC1, hENT1, dCK, 5’-NT, CDA, RRM1 e RRM2 in 85 campioni isolati con microdissezione da biopsie provenienti dai tessuti polmonari normali o tumorali o dalle metastasi linfonodali. Le analisi di questi tessuti hanno mostrato differenze significative per i pattern di espressione genica di diversi determinanti molecolari potenzialmente utile nel predire l’efficacia di gemcitabina/cisplatino e per personalizzare i trattamenti in pazienti affetti da cancro. In conclusione, l’evoluzione delle tecniche di biologia molecolare promossa dagli studi di farmacogenetica racchiude in sè notevoli potenzialità per quanto concerne l’ideazione di nuovi protocolli terapeutici. Identificando le caratteristiche genotipiche e i livelli di espressione geniche di determinanti molecolari implicati nella risposta ai farmaci potremmo infatti predisporre delle mappe di chemiosensibilità-chemioresistenza per ciascun paziente, nell’ottica di approntare di volta in volta le più appropriate terapie antitumorali in base alle caratteristiche genetiche del paziente e della sua patologia neoplastica

TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout

AbstractAcute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H2O2), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1+/+) or TRPA1-deficient (Trpa1−/−) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H2O2 generation, interleukin-1β release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H2O2 in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H2O2-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H2O2 mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H2O2 production as potential targets for treatment of acute gout attacks

Genetic association between bipolar disorder and 524A>C (Leu133Ile) polymorphism of CNR2gene, encoding for CB2 cannabinoid receptor. Interaction between Pum2 and Stau1 RNA-binding proteins and their role in behavior.

Genetic association between bipolar disorder and 524A>C (Leu133Ile) polymorphism of CNR2gene, encoding for CB2 cannabinoid receptor (abstract). Bipolar Disorder, an illness that affects an estimated 2.3 million American adults, has been characterized in many different ways. The original diagnosis of ―manic-depressive insanity,‖ described by Emil Kraepelin in the 1899 edition of Clinical Psychiatry, has evolved through the years to the current classification system of four subtypes: Bipolar I Disorder, Bipolar II Disorder, Cyclothymic Disorder and Bipolar Disorder Not Otherwise Specified. The primary mood disturbance in Bipolar I Disorder is either mania or a mixed episode and it is usually accompanied by episodes of depression while the primary mood disturbance in Bipolar II disorder is depression and it is accompanied by at least one episode of a mild form of mania called hypomania. Instead, an individual with Cyclothymic Disorder cycles between periods of hypomanic symptoms and periods of depressive Symptoms. The hypomanic symptoms are never severe enough to be considered a manic episode and the depressive symptoms are never severe enough to be considered a depressive episode. BD Not Otherwise Specified captures all of the other variants of the disease that do not fit nearly into one of the above categories. The etiology of BD is very complex. A lot of various biological system are involved in neurobiology of this disorder impairing function or just changing in morphology. The best characterized neurotransmitter systems involved in pathophysiology of BD are dopaminergic and serotononinergic, but also GABAergic and glutamatergic (Manji et al., 2003). More recently also the ECS has been identified as an important target of several psychiatric disorders including the bipolar one. In the past a lot of studies have investigated more the role of CBR1 in CNS than CBR2 because the expression of this latter receptor was considered only at microglia levels; recently the discovery of mRNA of the isoforms CBR2A in very important regions of human brain such as caudate, amygdale, hippocampus, cerebellum, nucleus accumbens, putamen and cortex (Liu et al., 2009) could permit to understand better the role of CBR2 not only in nuroinflammation process but also in neurotransmission and so also in depression and related disorders, as is BD. Then, the important role of CBR2 as mediator of PIM and the evidences that a lot of bipolar patients suffer of obesity, insulin resistance, arthritis, pain and cardiovascular illness, all pathologies correlated to alteration of CBR2, suggest that this CBR could really interfere with the pathology of BD. 34 So, in this study we tested the hypothesis that genetic variants of CNR2 might be associated with BD. In particular, by means of a case–control study the frequencies of three missense SNPs of CNR2, namely rs2501432 (315A>G; Arg63Gln), rs41311993 (524C>A; Leu133Ile) and rs2229579 (1073C>T; Tyr316His) (Fig.12) have been assessed in a sample of 80 BD patients and 160 healthy control.The comparison between patients and healthy group shows that allele frequencies were significantly different for CNR2 524C>A (p(χ2) =0.001) while there was no significant difference for CNR2 315A>G (p(χ2) =0.15) or1073C>T (p(χ2) =0.21). The CNR2 524C>A substitution leads to the aminoacid change Leu133Ile which has been suggested to influence the stability and/or functionality of the CBR2. In fact, Leu133 provides a hydrophobic residue involved in an intrachain bond in the transmembrane domains and its presence is thought to be crucial for the stability of the receptor and may influence the receptor-G protein coupling (Xie et al., 2003). Interaction between Pum2 and Stau1 RNA-binding proteins and their role in behavior (abstract). Many large or morphologically complex cells compartmentalize information by targeting either mRNAs or proteins to specific domains and maintain the localization of these molecules over time. These distinct cellular domains can function to determine cell polarity, define embryonic axes, or contribute to cellular memory. Although much is understood about how proteins move and are targeted in cells, our understanding of the mechanisms of RNA transport and localization are at an early stage. In neurobiology, the importance of these mechanisms for synaptic plasticity has recently become apparent. In this study, I have focused my attention on two RNA-binding proteins that are associated with impairments in learning and memory, in dendritic spine morphogenesis and in other aspects involved in synaptic plasticity: Stau1 and Pum2. Since the Stau/Pum pathway has been shown to be involved in long-term memory in Drosophila by Dubnau and colleagues, these two genes appear fundamental for memory formation (Dubnau et al., 2003). I have investigated how a gene trap mutation in one of these two genes could interfere with the expression of the other protein and which kind of effects these alteration could have in behavior features as response to a new environment and condition. Using transgenic mice for both proteins, Stau1tm1Apa and Pum2XE77 mice, I have shown that a gene trap mutation in one of these RNA-binding proteins changes not only the level of the disrupted protein, but also of the other one indicating a link between Stau1 and Pum2. To better understand these data I conducted behavioral tests for Stau1tm1Apa and Pum2XE77 mice analyzing several parameters to identify common and different features in behavior between the two transgenic mice groups. Our results are still preliminary and further studies, extending the sample number and the type of behavior test will be conducted in the future to validate the results presented here. Regardless, these results led me to conclude that the Stau/Pum pathway should be better investigated for its role in memory processes including the mechanism, which regulate the expression levels of these two proteins

In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach

AbstractDifferent optical imaging techniques have been developed to study neuronal activity with the goal of deciphering the neural code underlying neurophysiological functions. Because of several constraints inherent in these techniques as well as difficulties interpreting the results, the majority of these studies have been dedicated more to sensory modalities than to the spontaneous activity of the central brain. Recently, a novel bioluminescence approach based on GFP–aequorin (GA) (GFP: Green fluorescent Protein), has been developed, allowing us to functionally record in-vivo neuronal activity. Taking advantage of the particular characteristics of GA, which does not require light excitation, we report that we can record induced and/or the spontaneous Ca2+-activity continuously over long periods. Targeting GA to the mushrooms-bodies (MBs), a structure implicated in learning/memory and sleep, we have shown that GA is sensitive enough to detect odor-induced Ca2+-activity in Kenyon cells (KCs). It has been possible to reveal two particular peaks of spontaneous activity during overnight recording in the MBs. Other peaks of spontaneous activity have been recorded in flies expressing GA pan-neurally. Similarly, expression in the glial cells has revealed that these cells exhibit a cell-autonomous Ca2+-activity. These results demonstrate that bioluminescence imaging is a useful tool for studying Ca2+-activity in neuronal and/or glial cells and for functional mapping of the neurophysiological processes in the fly brain. These findings provide a framework for investigating the biological meaning of spontaneous neuronal activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium

The blockade of transient receptor potential ankirin 1 (TRPA1) signalling mediates antidepressant- and anxiolytic-like actions in mice

Transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) are involved in many biological processes, including nociception and hyperalgesia. Whereas the involvement of TRPV1 in psychiatric disorders such as anxiety and depression has been reported, little is known regarding the role of TRPA1 in these conditions