Nuovi aspetti biochimici e funzionali di due enzimi glutatione-dipendenti: γ-glutamil transferasi e glutatione transferasi omega

A) Ruolo della γ-glutamiltransferasi (GGT) nell'uptake cellulare dell'acido ascorbico. L'attivazione della GGT causa sia in sistemi in vitro acellulari che in linee tumorali con diversa attività di GGT, l'ossidazione dell'AA a DHA, insieme con la nota idrolisi del GSH; tale ossidazione viene inibita sia dal complesso serina/acido borico (SEB), inibitore competitivo della GGT, sia dalla presenza degli enzimi superossido dismutasi e catalasi nel mezzo di incubazione. In particolare la stimolazione dell'attività GGTasica nel clone 60 (linea con piu' alta attività GGT) ha come effetto principale un forte aumento dell'ossidazione dell'AA extracellulare e, contemporaneamente, un aumento significativo della concentrazione dell'AA intracellulare contrariamente al clone 21(bassa attività GGT). Incrementando l'attività GGTasica del clone 21 mediante transfezione transiente con il cDNA della GGT umana, le cellule acquisiscono una capacità di ossidare l'AA extracellulare e di aumentare i livelli intracellulari di vitamina C in maniera paragonabile a quella del clone 60. Abbiamo valutato il possibile coinvolgimento dei trasportatori GLUT nella captazione del DHA, ed la captazione di vitamina C é significativamente ridotta sia in presenza di elevate concentrazioni di glucosio, il normale substrato dei GLUT, sia in presenza di citocalasina B, inibitore non competitivo dei GLUT. Sulla base di questi dati possiamo dunque affermare che la GGT può svolgere un'azione ascorbato ossidasica nell'ambiente extracellulare, consentendo l'uptake cellulare di vitamina C sotto forma di DHA. B) Traslocazione nucleare della GSTO1 come marker di progressione tumorale nell'Esofago di Barrett.Sono stati esaminati 46 pazienti, di questi 44 presentavano Esofago di Barrett (EB), mentre 2 mostravano un adenocarcinoma conclamato. Tra i pazienti con EB, 22/44 non mostravano displasia, 7/44 mostravano diplasia di basso grado (DBG), 9/44 displasia di alto grado (DAG). Su 6/44 non é stato possibile fare diagnosi certa di displasia e pertanto sono stati classificati come "indeterminati per la displasia". Tramite tecniche di immunoistochimica, abbiamo evidenziato la localizzazione della GSTO1 a livello della mucosa metaplastica di Barrett distinguendo localizzazione esclusivamente citoplasmatica, esclusivamente nucleare o diffusa (sia nucleare che citoplasmatica).I casi senza displasia mostravano una localizzazione prevalentemente citoplasmatica, al contrario tutti i casi con chiara displasia, sia di basso grado sia di alto grado, mostravano una localizzazione prevalentemente nucleare della GSTO. La prevalenza della localizzazione nucleare tra i casi senza displasia e quelli con diplasia é significativamente diversa (p< 0,0001).I casi non definiti per la displasia, mostravano colorazione nucleare, citosolica e diffusa. Dei 2 casi di adenocarcinoma, uno mostrava prevalente localizzazione nucleare. In conclusione questi dati ci indicano che la traslocazione nucleare della GSTO1 può essere un marker di diplasia nell'Esofago di Barrett. C) Meccanismi d'espressione delle GSTO in linee cellulari umane. a) Trattamento con TNFα: un'aumentata espressione della GSTO valutata tramite immunoblotting e RT-PCR ; in particolare si osserva che il trattamento con TNFα induce l'aumento del mRNA della sola GSTO1. Dato che la stimolazione con TNF-α determina l'attivazione del fattore nucleare NFkB valutata tramite EMSA (Electrophoretic Mobility Shift Assay), esperimenti con il partenolide (inibitore di NFkB), hanno mostrato che l'inibizione dell'attivazione di NFkB blocca anche l'aumentata espressione di GSTO1 indotta dal TNF-α. Questi dati fanno pertanto supporre che il gene per la GSTO1 faccia parte del pattern di geni la cui trascrizione è regolata da NFkB. b) Effetto dell'aumento della densità cellulare:Il modello ad alta densità è stato ottenuto tramite due diversi approcci: 1) analisi nel tempo (ogni 24 ore a partire dalla semina fino a 96 ore) di campioni con lo stesso numero iniziale di cellule; 2) analisi nel medesimo tempo, ovvero dopo 24 ore di crescita, di campioni di cellule seminate a diverse densità.Tale studio è stato condotto su una linea di melanoma umano (clone 21), un secondo clone della linea di melanoma (clone 60) e le HeLa. In tutti i casi si è osservato che ad un'aumentata densità cellulare corrisponde un notevole incremento delle GSTO valutato tramite Immunoblotting e RT-PCR. In particolare l'incremento riguarda lo mRNA sia della GSTO1 che della GSTO2, anche se la prima appare più coinvolta. Abbiamo valutato i possibili meccanismi coinvolti nella modulazione dell'espressione di proteine da parte della densità cellulare tra cui la possibilità dell'instaurarsi delle connessioni tra cellule o tra cellule e substrato, delle variazioni del ciclo cellulare, dell'ipossia pericellulare, e della presenza di un fattore diffusibile. Ciascuno di questi casi ha dato esito negativo. Abbiamo infine voluto verificare l'ipotesi che la sovraespressione densità-dipendente delle GSTO sia dovuta a una produzione extracellulare di ROS. In esperimenti preliminari, trattando cellule HeLa ad alta densità con superossido dismutasi (SOD), si inibisce completamente la sovraespressione delle GSTO, riportandola ai valori basali osservati nelle colture a bassa densità

Iron Metabolism in Liver Cancer Stem Cells

Cancer stem cells (CSC) which have been identified in several tumors, including liver cancer, represent a particular subpopulation of tumor cells characterized by properties similar to those of adult stem cells. Importantly, CSC are resistant to standard therapies, thereby leading to metastatic dissemination and tumor relapse. Given the increasing evidence that iron homeostasis is deregulated in cancer, here we describe the iron homeostasis alterations in cancer cells, particularly in liver CSC. We also discuss two paradoxically opposite iron manipulation-strategies for tumor therapy based either on iron chelation or iron overload-mediated oxidant production leading to ferroptosis. A better understanding of iron metabolism modifications occurring in hepatic tumors and particularly in liver CSC cells may offer new therapeutic options for this cancer, which is characterized by increasing incidence and unfavorable prognosis

Plasma membrane gamma-glutamyltransferase activity facilitates the uptake of vitamin C in melanoma cells.

Adequate cellular transport of ascorbic acid (AA) and its oxidation product dehydroascorbate (DHA) is assured through specific carriers. It was shown that vitamin C is taken up as DHA by most cell types, including cancer cells, via the facilitative GLUT transporters. Thus, AA oxidation to DHA can be considered a mechanism favoring vitamin C uptake and intracellular accumulation. We have investigated whether such an AA-oxidizing action might be provided by plasma membrane g-glutamyltransferase (GGT), previously shown to function as an autocrine source of prooxidants. The process was studied using two distinct human metastatic melanoma clones. It was observed that the Me665/2/60 clone, expressing high levels of membrane GGT activity, was capable of effecting the oxidation of extracellular AA, accompanied by a marked increase of intracellular AA levels. The phenomenon was not observed with Me665/2/21 cells, possessing only traces of membrane GGT. On the other hand, AA oxidation and stimulation of cellular uptake were indeed observed after transfection of 2/21 cells with cDNA coding for GGT. The mechanism of GGTmediated AA oxidation was investigated in acellular systems, including GGT and its substrate glutathione. The process was observed in the presence of redox-active chelated iron(II) and of transferrin or ferritin, i.e., two physiological iron sources. Thus, membrane GGT activity—often expressed at high levels in human malignancies—can oxidize extracellular AA and promote its uptake efficiently

Immunohistochemical localization of histidine-rich glycoprotein in human skeletal muscle: preferential distribution of the protein at the sarcomeric I-band

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that is synthesized by parenchymal liver cells. Using Western blot analysis and immunoperoxidase techniques, we have previously shown the presence of HRG in human skeletal muscle. This paper reports the results of immunofluorescence experiments carried out on sections of human normal skeletal muscle biopsies to investigate the subcellular localization of HRG. The HRG localization was also compared with that of skeletal muscle AMP deaminase (AMPD1), since we have previously described an association of the enzyme with the protein. The obtained results give evidence for a preferential localization of HRG at the I-band level, where it shows the same distribution of actin and where AMPD1 is present in major concentration

Lacking P2X7-receptors protects substantia nigra dopaminergic neurons and hippocampal-related cognitive performance from the deleterious effects of high-fat diet exposure in adult male mice

BackgroundDietary fat consumption, involved in the pathogenesis of insulin resistance and impaired glucose metabolism, is linked with decline in cognitive functions, dementia, and development of Parkinson’s disease and Alzheimer’s disease. Mature IL-1β, requiring the activation of the P2X7 receptor (P2X7R)-inflammasome complex, is an important mediator of neuroinflammation. The aim of the study was to test whether P2X7R activation might interfere with systemic and cerebral metabolic homeostasis.MethodsWe treated WT and P2X7R KO mice with a high-fat diet (HFD) for 16 weeks, evaluating the effects on the Substantia Nigra and Hippocampus, target areas of damage in several forms of cognitive impairment.ResultsHFD-treated WT and P2X7R KO mice showed a different brain mRNA profile of Insulin and Igf-1, with these genes and relative receptors, more expressed in KO mice. Unlike P2X7R KO mice, WT mice treated with HFD displayed a diameter reduction in dopaminergic neurons in the Substantia Nigra, accompanied by an increased IBA1 expression in this area; they also showed poor performances during Y-Maze and Morris Water Maze, tasks involving Hippocampus activity. Conversely, Parkin, whose reduction might promote neuronal cell death, was increased in the brain of P2X7R KO animals.ConclusionWe report for the first time that HFD induces damage in dopaminergic neurons of the Substantia Nigra and a Hippocampus-related worse cognitive performance, both attenuated in the absence of P2X7R. The involved mechanisms might differ in the two brain areas, with a predominant role of inflammation in the Substantia Nigra and a metabolic derangement in the Hippocampus

Growth Hormone Is Necessary for the p53-Mediated, Obesity-Induced Insulin Resistance in Male C57BL/6J × CBA Mice

Insulin resistance is a key marker of both obesity and GH excess. The purpose of the study was to assess the role of GH on p53-mediated insulin resistance of male mice with obesity due to a high-fat diet. C57BL/6J CBA male mice fed on a high-fat diet (Obe) were studied; male mice fed a normal diet (Lean) or transgenic mice for bovine GH under the same genetic background (Acro) served as controls. The convergence of p53 and GH pathways was evaluated by Western blot. Obe mice had insulin resistance, which was sustained by a selective increased expression of p53 in adipose tissue. Normal insulin sensitivity was restored, and adipose p53 expression normalized when the GH pathway was blocked. Only the adipose p53 expression was sensitive to the GH blockage, which occurred through the p38 pathway. Adipose tissue of Obe mice had a coordinate overexpres- sion of suppressors of cytokine signal 1–3 and signal transducers and activators of transcrip- tion-1, -3, and -5b, not different from that of Acro mice, suggesting an increased sensitivity of adipose tissue to GH. On the contrary, Lean mice were unaffected by changes of GH action. GH seems to be necessary for the increased adipose p53 expression and for insulin resistance of obese mice

Proteomic profiling of extracellular vesicles in synovial fluid and plasma from Oligoarticular Juvenile Idiopathic Arthritis patients reveals novel immunopathogenic biomarkers

IntroductionNew early low-invasive biomarkers are demanded for the management of Oligoarticular Juvenile Idiopathic Arthritis (OJIA), the most common chronic pediatric rheumatic disease in Western countries and a leading cause of disability. A deeper understanding of the molecular basis of OJIA pathophysiology is essential for identifying new biomarkers for earlier disease diagnosis and patient stratification and to guide targeted therapeutic intervention. Proteomic profiling of extracellular vesicles (EVs) released in biological fluids has recently emerged as a minimally invasive approach to elucidate adult arthritis pathogenic mechanisms and identify new biomarkers. However, EV-prot expression and potential as biomarkers in OJIA have not been explored. This study represents the first detailed longitudinal characterization of the EV-proteome in OJIA patients.MethodsFourty-five OJIA patients were recruited at disease onset and followed up for 24 months, and protein expression profiling was carried out by liquid chromatography-tandem mass spectrometry in EVs isolated from plasma (PL) and synovial fluid (SF) samples.ResultsWe first compared the EV-proteome of SF vs paired PL and identified a panel of EV-prots whose expression was significantly deregulated in SF. Interaction network and GO enrichment analyses performed on deregulated EV-prots through STRING database and ShinyGO webserver revealed enrichment in processes related to cartilage/bone metabolism and inflammation, suggesting their role in OJIA pathogenesis and potential value as early molecular indicators of OJIA development. Comparative analysis of the EV-proteome in PL and SF from OJIA patients vs PL from age/gender-matched control children was then carried out. We detected altered expression of a panel of EV-prots able to differentiate new-onset OJIA patients from control children, potentially representing a disease-associated signature measurable at both the systemic and local levels with diagnostic potential. Deregulated EV-prots were significantly associated with biological processes related to innate immunity, antigen processing and presentation, and cytoskeleton organization. Finally, we ran WGCNA on the SF- and PL-derived EV-prot datasets and identified a few EV-prot modules associated with different clinical parameters stratifying OJIA patients in distinct subgroups.DiscussionThese data provide novel mechanistic insights into OJIA pathophysiology and an important contribution in the search of new candidate molecular biomarkers for the disease

Cholangiocarcinoma 2020: the next horizon in mechanisms and management

[EN] Cholangiocarcinoma (CCA) includes a cluster of highly heterogeneous biliary malignant tumours that can arise at any point of the biliary tree. Their incidence is increasing globally, currently accounting for ~15% of all primary liver cancers and ~3% of gastrointestinal malignancies. The silent presentation of these tumours combined with their highly aggressive nature and refractoriness to chemotherapy contribute to their alarming mortality, representing ~2% of all cancer-related deaths worldwide yearly. The current diagnosis of CCA by non- invasive approaches is not accurate enough, and histological confirmation is necessary. Furthermore, the high heterogeneity of CCAs at the genomic, epigenetic and molecular levels severely compromises the efficacy of the available therapies. In the past decade, increasing efforts have been made to understand the complexity of these tumours and to develop new diagnostic tools and therapies that might help to improve patient outcomes. In this expert Consensus Statement, which is endorsed by the European Network for the Study of Cholangiocarcinoma, we aim to summarize and critically discuss the latest advances in CCA, mostly focusing on classification, cells of origin, genetic and epigenetic abnormalities, molecular alterations, biomarker discovery and treatments. Furthermore, the horizon of CCA for the next decade from 2020 onwards is highlightedJ.M.B. received EASL Registry Awards 2016 and 2019 (European CCA Registry, ENS-CCA). J.M.B. and M.J.P. were supported by: the Spanish Ministry of Economy and Competitiveness (J.M.B.: FIS PI12/00380, FIS PI15/01132, FIS PI18/01075 and Miguel Servet Programme CON14/00129; M.J.P.: FIS PI14/00399, FIS PI17/00022 and Ramon y Cajal Programme RYC-2015-17755, co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER)); ISCIII CIBERehd; “Diputación Foral de Gipuzkoa” (J.M.B: DFG15/010, DFG16/004), and BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/BD); the Department of Health of the Basque Country (M.J.P.: 2015111100; J.M.B.: 2017111010), and “Fundación Científica de la Asociación Española Contra el Cancer” (AECC Scientific Foundation) (J.M.B.). J.M.B. and J.W.V. were supported by the European Commission Horizon 2020 programme (ESCALON project 825510). The laboratory of J.B.A. is supported by competitive grants from the Danish Medical Research Council, the Danish Cancer Society, and the Novo Nordisk and A.P. Møller Foundations. J.J.G.M. and R.I.R.M. were supported by the Carlos III Institute of Health, Spain (PI16/00598 and PI18/00428) and were co-financed by the European Regional Development Fund. J.M.B. and J.J.G.M. were supported by the Ministry of Science and Innovation, Spain (SAF2016-75197-R), and the “Asociación Española Contra el Cancer”, Spain (AECC-2017). R.I.R.M. was supported by the “Centro Internacional sobre el Envejecimiento”, Spain (OLD-HEPAMARKER, 0348-CIE-6-E). A.L. received funding from the Christie Charity. M.M. was supported by the Università Politecnica delle Marche, Ancona, Italy (040020_R.SCIENT.A_2018_MARZIONI_M_STRATEGICO_2017). M.S. was supported by the Yale Liver Center Clinical and Translational Core and the Cellular and Molecular Core (DK034989 Silvio O. Conte Digestive Diseases Research Center). C.C. is supported by grants from INSERM, Université de Rennes, INCa, and ITMO Cancer AVIESAN dans le cadre du Plan Cancer (Non-coding RNA in Cancerology: Fundamental to Translational), Ligue Contre le Cancer and Région Bretagne. J.Bruix was supported by grants from Instituto de Salud Carlos III (PI18/00763), AECC (PI044031) and WCR (AICR) 16-0026. A.F. was supported by grants from ISCIII (PI13/01229 and PI18/00542). CIBERehd is funded by the Instituto de Salud Carlos III. V.C., D.M., J. Bridgewater and P.I. are members of the European Reference Network - Hepatological Diseases (ERN RARE-LIVER). J.M.B. is a collaborator of the ERN RARE-LIVER

Criteria for preclinical models of cholangiocarcinoma:scientific and medical relevance

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA