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1.
Hepatology ; 2021 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-34743371

RESUMO

BACKGROUND AND AIMS: Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein-coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFß receptor type I (TGFßRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. APPROACH AND RESULTS: Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2-/- ) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2-/- mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin's interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFßR1 signaling, which was reduced by loss of MT1. CONCLUSIONS: Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFßR1 activation. Blocking GPR50/TGFßR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.

2.
Biomaterials ; 277: 121067, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34517276

RESUMO

Epithelial cell therapies have been at an impasse because of inefficient methods of transplantation to solid organs. Patch grafting strategies were established enabling transplantation of ≥107th organoids/patch of porcine GFP+ biliary tree stem/progenitors into livers of wild type hosts. Grafts consisted of organoids embedded in soft (~100 Pa) hyaluronan hydrogels, both prepared in serum-free Kubota's Medium; placed against target sites; covered with a silk backing impregnated with more rigid hyaluronan hydrogels (~700 Pa); and use of the backing to tether grafts with sutures or glue to target sites. Hyaluronan coatings (~200-300 Pa) onto the serosal surface of the graft served to minimize adhesions with neighboring organs. The organ's clearance of hyaluronans enabled restoration of tissue-specific paracrine and systemic signaling, resulting in return of normal hepatic histology, with donor parenchymal cells uniformly integrated amidst host cells and that had differentiated to mature hepatocytes and cholangiocytes. Grafts containing donor mature hepatocytes, partnered with endothelia, and in the same graft biomaterials as for stem/progenitor organoids, did not engraft. Engraftment occurred if porcine liver-derived mesenchymal stem cells (MSCs) were co-transplanted with donor mature cells. RNA-seq analyses revealed that engraftment correlated with expression of matrix-metalloproteinases (MMPs), especially secreted isoforms that were found expressed strongly by organoids, less so by MSCs, and minimally, if at all, by adult cells. Engraftment with patch grafting strategies occurred without evidence of emboli or ectopic cell distribution. It was successful with stem/progenitor organoids or with cells with a source(s) of secreted MMP isoforms and offers significant potential for enabling cell therapies for solid organs.


Assuntos
Fígado , Organoides , Animais , Diferenciação Celular , Hepatócitos , Células-Tronco , Suínos
3.
Hepatology ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34543480

RESUMO

BACKGROUND & AIMS: Non-anastomotic biliary strictures (NAS) are a major cause of morbidity after orthotopic liver transplantation (OLT). Although ischemic injury of peribiliary glands (PBGs) and peribiliary vascular plexus (PVP) during OLT has been associated with the later development of NAS, the exact underlying mechanisms remain unclear. We hypothesized that bile ducts of patients with NAS suffer from ongoing biliary hypoxia and lack of regeneration from PBG stem/progenitor cells. APPROACH & RESULTS: Forty-two patients, requiring retransplantation for either NAS (n=18), hepatic artery thrombosis (HAT; n=13) or non-biliary graft failure (controls; n=11), were included in this study. Histomorphologic analysis of perihilar bile ducts was performed to assess differences in markers of cell proliferation and differentiation in PBGs, microvascular density, and hypoxia. In addition, isolated human biliary tree stem cells (hBTSCs) were used to examine exo-metabolomics during in vitro differentiation toward mature cholangiocytes. Bile ducts of patients with NAS or HAT had significantly reduced indices of PBG mass, cellular proliferation and differentiation (mucus production, secretin receptor expression, primary cilia), reduced microvascular density, and increased PBG apoptosis and hypoxia marker expression, compared to controls. Metabolomics of hBTSCs during in vitro differentiation toward cholangiocytes revealed a switch from a glycolytic to oxidative metabolism, indicating the need for oxygen. CONCLUSIONS: NAS are characterized by a microscopic phenotype of chronic biliary hypoxia due to loss of microvasculature, resulting in reduced proliferation and differentiation of PBG stem/progenitor cells into mature cholangiocytes. These findings suggest that persistent biliary hypoxia is a key mechanism underlying the development of NAS after OLT.

4.
J S Afr Vet Assoc ; 92(0): e1-e8, 2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34476958

RESUMO

Chemical immobilisation is essential for veterinarians to perform medical procedures in wild African ungulates. Potent opioids combined with neuroleptic drugs are most often used for this purpose. The present study aimed at comparing the quality of immobilisation and effects on physiological variables between a high (high etorphine-azaperone [HE]: 0.09 mg kg-1) and low etorphine dose (low etorphine-azaperone [LE]: 0.05 mg kg-1), both combined with azaperone (0.35 mg kg-1), in 12 adult female boma-acclimatised blesbok. It was hypothesised that a reduction in etorphine's dose in combination with azaperone would result in less cardiorespiratory impairment but likely worsen the quality of immobilisation. Both treatments resulted in rapid induction and recovery times. Overall inter-treatment differences occurred in pulse rate (HE and LE: 52 ± 15 and 44 ± 11 beats minute-1, p 0.0001), respiratory rate (HE and LE: 15 ± 4 and 17 ± 4 breaths minute-1, p 0.006), partial pressure of exhaled carbon dioxide (HE and LE: 62.0 ± 5.0 and 60.0 ± 5.6 millimetre of mercury [mmHg], p 0.028) and arterial carbon dioxide (HE and LE: 58.0 ± 4.5 and 55.0 ± 3.9 mmHg, p 0.002). Both HE and LE led to bradycardia, hypertension and marked hypoxia to a similar extent. Furthermore, quality of induction, immobilisation and recovery were similar in both treatments. The role of azaperone in the development of cardiorespiratory compromise and gas exchange impairment that occurred when these combinations were used is still unclear. Further studies are recommended to elucidate drug- and dose-specific physiological effects in immobilised antelope.


Assuntos
Antílopes , Azaperona/farmacologia , Etorfina/farmacologia , Hipnóticos e Sedativos/farmacologia , Imobilização/veterinária , Animais , Combinação de Medicamentos , Feminino , Imobilização/métodos , Monitorização Fisiológica/veterinária
5.
J Hepatol ; 75(6): 1377-1386, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34329660

RESUMO

BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

6.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298894

RESUMO

(1) The high-fat diet (HFD) of western countries has dramatic effect on the health of several organs, including the digestive tract, leading to the accumulation of fats that can also trigger a chronic inflammatory process, such as that which occurs in non-alcohol steatohepatitis. The effects of a HFD on the small intestine, the organ involved in the absorption of this class of nutrients, are still poorly investigated. (2) To address this aspect, we administered a combined HFD with sucrose (HFD w/Suc, fat: 58% Kcal) regimen (18 months) to mice and investigated the morphological and molecular changes that occurred in the wall of proximal tract of the small intestine compared to the intestine of mice fed with a standard diet (SD) (fat: 18% Kcal). (3) We found an accumulation of lipid droplets in the mucosa of HFD w/Suc-fed mice that led to a disarrangement of mucosa architecture. Furthermore, we assessed the expression of several key players involved in lipid metabolism and inflammation, such as perilipin, leptin, leptin receptor, PI3K, p-mTOR, p-Akt, and TNF-α. All these molecules were increased in HFD mice compared to the SD group. We also evaluated anti-inflammatory molecules like adiponectin, adiponectin receptor, and PPAR-γ, and observed their significant reduction in the HFD w/Suc group compared to the control. Our data are in line with the knowledge that improper eating habits present a primary harmful assault on the bowel and the entire body's health. (4) These results represent a promising starting point for future studies, helping to better understand the complex and not fully elucidated spectrum of intestinal alterations induced by the overconsumption of fat.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/administração & dosagem , Sacarose na Dieta/efeitos adversos , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Adiponectina/metabolismo , Animais , Comportamento Alimentar/fisiologia , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Leptina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Obesidade/patologia , PPAR gama/metabolismo , Receptores para Leptina/metabolismo
7.
Int J Mol Sci ; 22(11)2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34198853

RESUMO

In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipase/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Hepatopatia Gordurosa não Alcoólica/genética , Adulto , Idoso , Feminino , Regulação da Expressão Gênica/genética , Hepacivirus/genética , Hepatócitos/virologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Gotículas Lipídicas/virologia , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/virologia , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos
8.
Cell Death Dis ; 12(7): 686, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34238932

RESUMO

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP-/- than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability.


Assuntos
Autofagia , Proteína Beclina-1/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Fibroblastos/metabolismo , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Fibroblastos/patologia , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Ubiquitinação
10.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070643

RESUMO

Cholangiocarcinomas (CCAs) are heterogeneous biliary tract malignancies with dismal prognosis, mainly due to tumor aggressiveness, late diagnosis, and poor response to current therapeutic options. High-throughput technologies have been used as a fundamental tool in unveiling CCA molecular landscape, and several molecular classifications have been proposed, leading to various targeted therapy trials. In this review, we aim to analyze the critical issues concerning the status of precision medicine in CCA, discussing molecular signatures and clusters, related to both anatomical classification and different etiopathogenesis, and the latest therapeutic strategies. Furthermore, we propose an integrated approach comprising the CCA molecular mechanism, pathobiology, clinical and histological findings, and treatment perspectives for the ultimate purpose of improving the methods of patient allocations in clinical trials and the response to personalized therapies.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Terapia de Alvo Molecular , Medicina de Precisão , Neoplasias dos Ductos Biliares/classificação , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/terapia , Colangiocarcinoma/classificação , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/terapia , Humanos , Microambiente Tumoral
11.
Front Med (Lausanne) ; 8: 610189, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937276

RESUMO

The intestinal extracellular matrix (ECM) represents a complex network of proteins that not only forms a support structure for resident cells but also interacts closely with them by modulating their phenotypes and functions. More than 300 molecules have been identified, each of them with unique biochemical properties and exclusive biological functions. ECM components not only provide a scaffold for the tissue but also afford tensile strength and limit overstretch of the organ. The ECM holds water, ensures suitable hydration of the tissue, and participates in a selective barrier to the external environment. ECM-to-cells interaction is crucial for morphogenesis and cell differentiation, proliferation, and apoptosis. The ECM is a dynamic and multifunctional structure. The ECM is constantly renewed and remodeled by coordinated action among ECM-producing cells, degrading enzymes, and their specific inhibitors. During this process, several growth factors are released in the ECM, and they, in turn, modulate the deposition of new ECM. In this review, we describe the main components and functions of intestinal ECM and we discuss their role in maintaining the structure and function of the intestinal barrier. Achieving complete knowledge of the ECM world is an important goal to understand the mechanisms leading to the onset and the progression of several intestinal diseases related to alterations in ECM remodeling.

12.
Oncogene ; 40(22): 3799-3814, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33958721

RESUMO

Despite the development of new targeted and immune therapies, the prognosis of metastatic melanoma remains bleak. Therefore, it is critical to better understand the mechanisms controlling advanced melanoma to develop more effective treatment regimens. Hedgehog/GLI (HH/GLI) signaling inhibitors targeting the central pathway transducer Smoothened (SMO) have shown to be clinical efficacious in skin cancer; however, several mechanisms of non-canonical HH/GLI pathway activation limit their efficacy. Here, we identify a novel SOX2-BRD4 transcriptional complex driving the expression of GLI1, the final effector of the HH/GLI pathway, providing a novel mechanism of non-canonical SMO-independent activation of HH/GLI signaling in melanoma. Consistently, we find a positive correlation between the expression of GLI1 and SOX2 in human melanoma samples and cell lines. Further, we show that combined targeting of canonical HH/GLI pathway with the SMO inhibitor MRT-92 and of the SOX2-BRD4 complex using a potent Proteolysis Targeted Chimeras (PROTACs)-derived BRD4 degrader (MZ1), yields a synergistic anti-proliferative effect in melanoma cells independently of their BRAF, NRAS, and NF1 mutational status, with complete abrogation of GLI1 expression. Combination of MRT-92 and MZ1 strongly potentiates the antitumor effect of either drug as single agents in an orthotopic melanoma model. Together, our data provide evidence of a novel mechanism of non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex, and describe the efficacy of a new combinatorial treatment for a subset of melanomas with an active SOX2-BRD4-GLI1 axis.

13.
Blood Adv ; 5(10): 2467-2480, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33999145

RESUMO

Histone deacetylase inhibitors (HDACis) are antitumor agents with distinct efficacy in hematologic tumors. Pracinostat is a pan-HDACi with promising early clinical activity. However, similar to other HDACis, its activity as a single agent is limited. Diffuse large B-cell lymphoma (DLBCL) includes distinct molecular subsets or metabolically defined subtypes that rely in different ways on the B-cell receptor signaling pathway, oxidative phosphorylation, and glycolysis for their survival. The antitumor activity of pracinostat has not been determined in lymphomas. We performed preclinical in vitro activity screening of 60 lymphoma cell lines that included 25 DLBCLs. DLBCL cells belonging to distinct metabolic subtypes were treated with HDACis for 6 hours or 14 days followed by transcriptional profiling. DLBCL xenograft models enabled assessment of the in vivo antilymphoma activity of pracinostat. Combination treatments with pracinostat plus 10 other antilymphoma agents were performed. Western blot was used to assess acetylation levels of histone and nonhistone proteins after HDACi treatment. Robust antiproliferative activity was observed across all lymphoma histotypes represented. Focusing on DLBCL, we identified a low-sensitivity subset that almost exclusively consists of the oxidative phosphorylation (OxPhos)-DLBCL metabolic subtype. OxPhos-DLBCL cells also showed poorer sensitivity to other HDACis, including vorinostat. Transcriptomic analysis revealed fewer modulated transcripts but an enrichment of antioxidant pathway genes after HDACi treatment of OxPhos-DLBCLs compared with high-sensitivity B-cell receptor (BCR)-DLBCLs. Pharmacologic inhibition of antioxidant production rescued sensitivity of OxPhos-DLBCLs to pracinostat whereas BCR-DLBCLs were unaffected. Our study provides novel insights into the antilymphoma activity of pracinostat and identifies a differential response of DLBCL metabolic subtypes to HDACis.


Assuntos
Antineoplásicos , Linfoma Difuso de Grandes Células B , Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética
14.
Hepatology ; 74(3): 1496-1508, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33724515

RESUMO

BACKGROUND AND AIMS: Liver fibrosis holds a relevant prognostic meaning in primary biliary cholangitis (PBC). Noninvasive fibrosis evaluation using vibration-controlled transient elastography (VCTE) is routinely performed. However, there is limited evidence on its accuracy at diagnosis in PBC. We aimed to estimate the diagnostic accuracy of VCTE in assessing advanced fibrosis (AF) at disease presentation in PBC. APPROACH AND RESULTS: We collected data from 167 consecutive treatment-naïve PBC patients who underwent liver biopsy (LB) at diagnosis at six Italian centers. VCTE examinations were completed within 12 weeks of LB. Biopsies were scored by two blinded expert pathologists, according to the Ludwig system. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROCs) for AF (Ludwig stage ≥III). Effects of biochemical and clinical parameters on liver stiffness measurement (LSM) were appraised. The derivation cohort consisted of 126 patients with valid LSM and LB; VCTE identified patients with AF with an AUROC of 0.89. LSM cutoffs ≤6.5 and >11.0 kPa enabled to exclude and confirm, respectively, AF (negative predictive value [NPV] = 0.94; positive predictive value [PPV] = 0.89; error rate = 5.6%). These values were externally validated in an independent cohort of 91 PBC patients (NPV = 0.93; PPV = 0.89; error rate = 8.6%). Multivariable analysis found that the only parameter affecting LSM was fibrosis stage. No association was found with BMI and liver biochemistry. CONCLUSIONS: In a multicenter study of treatment-naïve PBC patients, we identified two cutoffs (LSM ≤6.5 and >11.0 kPa) able to discriminate at diagnosis the absence or presence, respectively, of AF in PBC patients, with external validation. In patients with LSM between these two cutoffs, VCTE is not reliable and liver biopsy should be evaluated for accurate disease staging. BMI and liver biochemistry did not affect LSMs.

15.
Front Cell Dev Biol ; 9: 629182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33614661

RESUMO

Oleic acid (OA) is a component of the olive oil. Beneficial health effects of olive oil are well-known, such as protection against liver steatosis and against some cancer types. In the present study, we focused on OA effects in hepatocellular carcinoma (HCC), investigating responses to OA treatment (50-300 µM) in HCC cell lines (Hep3B and Huh7.5) and in a healthy liver-derived human cell line (THLE-2). Upon OA administration higher lipid accumulation, perilipin-2 increase, and autophagy reduction were observed in HCC cells as compared to healthy cells. OA in the presence of 10% FBS significantly reduced viability of HCC cell lines at 300 µM through Alamar Blue staining evaluation, and reduced cyclin D1 expression in a dose-dependent manner while it was ineffective on healthy hepatocytes. Furthermore, OA increased cell death by about 30%, inducing apoptosis and necrosis in HCC cells but not in healthy hepatocytes at 300 µM dosage. Moreover, OA induced senescence in Hep3B, reduced P-ERK in both HCC cell lines and significantly inhibited the antiapoptotic proteins c-Flip and Bcl-2 in HCC cells but not in healthy hepatocytes. All these results led us to conclude that different cell death processes occur in these two HCC cell lines upon OA treatment. Furthermore, 300 µM OA significantly reduced the migration and invasion of both HCC cell lines, while it has no effects on healthy cells. Finally, we investigated autophagy role in OA-dependent effects by using the autophagy inducer torin-1. Combined OA/torin-1 treatment reduced lipid accumulation and cell death as compared to single OA treatment. We therefore concluded that OA effects in HCC cells lines are, at least, in part dependent on OA-induced autophagy reduction. In conclusion, we report for the first time an autophagy dependent relevant anti-cancer effect of OA in human hepatocellular carcinoma cell lines.

16.
Sci Rep ; 11(1): 2557, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510179

RESUMO

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.


Assuntos
Colangiocarcinoma/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Metformina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína Forkhead Box O3/metabolismo , Humanos , Camundongos , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos
17.
Stem Cell Rev Rep ; 17(2): 673-684, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33165749

RESUMO

All-trans retinoic acid (ATRA) promotes the development and the function of insulin producing cells and induces partial differentiation of pancreatic tumor cells. A number of evidences clearly indicate that the ATRA mediated signaling may have a substantial role in therapeutic approaches based on restoration of functional ß-cells. Among the proteins up-regulated by ATRA, Vav1 is involved in maturation and function of haematopoietic cells and is essential for retinoids induced differentiation of tumor promyelocytes. The presence of Vav1 in solid tissues, including pancreas, is considered ectopic and no role in the differentiation of human epithelial cells has so far been described. We demonstrated here that Vav1 sustains the maturation to ß-cells of the normal precursors human Biliary Tree Stem/progenitor Cells (hBTSCs) induced by a differentiation medium containing ATRA and that, in the mature normal pancreas, insulin-producing cells express variable levels of Vav1. Using pancreatic ductal adenocarcinoma (PDAC)-derived cells, we also revealed that the ATRA induced up-modulation of Vav1 is essential for the retinoid-induced trans-differentiation of neoplastic cells into insulin producing cells. The results of this study identify Vav1 as crucial molecule in ATRA induced maturation of insulin producing cells and suggest this protein as a marker for new strategies ended to restore functional ß-cells. Graphical abstract.

18.
Expert Opin Investig Drugs ; 30(4): 365-375, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33226854

RESUMO

Introduction: Cholangiocarcinoma (CCA) is a devastating liver tumor with a poor prognosis. While less than 50% of the patients with CCA may benefit from surgical resection, the rest undergoes chemotherapy with disappointing results (mean survival <2 years). Alternative pharmacological treatments are needed to improve the outcomes in patients with CCA.Areas covered: In this review, we discuss CCA-related (1) experimental systems used in preclinical studies; (2) pharmacological targets identified by genetic analysis; (3) results obtained in preliminary trials in human with their pros and cons; and (4) possible targeting of endocrinal modulation. A PubMed bibliographic search matching the term 'cholangiocarcinoma' with 'experimental model', 'preclinical model', 'genetic target', 'targeted therapy', 'clinical trial', or 'translational research' was conducted and manuscripts published between 2010 and 2020 were retrieved for reading and reviewing.Expert opinion: Several factors contribute to the translational gap between bench research and clinical practice in CCA. The tumor heterogeneity, lack of a preclinical model recapitulating the different features of CCA, and difficult patient enrollment in clinical trials are elements to consider for basic and clinical research in CCA. Establishment of international networks formed by experts in the field of CCA may improve future research and its translational findings on patients.


Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Terapia de Alvo Molecular , Animais , Antineoplásicos/administração & dosagem , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Desenvolvimento de Medicamentos , Humanos , Prognóstico , Taxa de Sobrevida , Pesquisa Médica Translacional
19.
Haematologica ; 105(11): 2584-2591, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33131247

RESUMO

Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematological cancers. Here, we report preclinical data on the anti-tumor activity of the first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination and validation experiments on in vivo models. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25/42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax, and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.


Assuntos
Antineoplásicos , Imunoconjugados , Linfoma , Anticorpos Monoclonais/farmacologia , Antígenos CD20 , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Linfoma/tratamento farmacológico , Rituximab/uso terapêutico
20.
Eur J Histochem ; 64(4)2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33131269

RESUMO

Cholangiocarcinoma (CCA) represents the second most common primary hepatic malignancy and originates from the neoplastic transformation of the biliary cells. The intrahepatic subtype includes two morpho-molecular forms: large-duct type intrahepatic CCA (iCCA) and small-duct type iCCA. Iron is fundamental for the cellular processes, contributing in tumor development and progression. The aim of this study was to evaluate iron uptake, storage, and efflux proteins in both lipopolysaccharide-inflamed small and large cholangiocytes as well as in different iCCA subtypes. Our results show that, despite an increase in interleukin-6 production by both small and large cholangiocytes, ferroportin (Fpn) was decreased only in small cholangiocytes, whereas transferrin receptor-1 (TfR1) and ferritin (Ftn) did not show any change. Differently from in vitro models, Fpn expression was increased in malignant cholangiocytes of small-duct type iCCA in comparison to large-duct type iCCA and peritumoral tissues. TfR1, Ftn and hepcidin were enhanced, even if at different extent, in both malignant cholangiocytes in comparison to the surrounding samples. Lactoferrin was higher in large-duct type iCCA in respect to small-duct type iCCA and peritumoral tissues. These findings show a different iron handling by inflamed small and large cholangiocytes, and small and large-duct type iCCA. The difference in iron homeostasis by the iCCA subtypes may have implications for the tumor management.


Assuntos
Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Ferro/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/metabolismo , Neoplasias dos Ductos Biliares/classificação , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Proteínas de Transporte de Cátions/metabolismo , Colangiocarcinoma/classificação , Colangiocarcinoma/patologia , Células Epiteliais/classificação , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Ferritinas/metabolismo , Hepcidinas/metabolismo , Humanos , Pessoa de Meia-Idade , Receptores da Transferrina/metabolismo
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