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The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell-cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.
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Neoplasias das Glândulas Suprarrenais , Feocromocitoma , Humanos , Glândulas Suprarrenais/metabolismo , Catecolaminas/metabolismo , Cromograninas/metabolismoRESUMO
Paget's disease (PDB) is a late-onset bone remodeling disorder with a broad spectrum of symptoms and complications. One of the most aggressive forms is caused by the P937R mutation in the ZNF687 gene. Although the genetic involvement of ZNF687 in PDB has been extensively studied, the molecular mechanisms underlying this association remain unclear. Here, we describe the first Zfp687 knock-in mouse model and demonstrate that the mutation recapitulates the PDB phenotype, resulting in severely altered bone remodeling. Through microcomputed tomography analysis, we observed that 8-month-old mutant mice showed a mainly osteolytic phase, with a significant decrease in the trabecular bone volume affecting the femurs and the vertebrae. Conversely, osteoblast activity was deregulated, producing disorganized bone. Notably, this phenotype became pervasive in 16-month-old mice, where osteoblast function overtook bone resorption, as highlighted by the presence of woven bone in histological analyses, consistent with the PDB phenotype. Furthermore, we detected osteophytes and intervertebral disc degeneration, outlining for the first time the link between osteoarthritis and PDB in a PDB mouse model. RNA sequencing of wild-type and Zfp687 knockout RAW264.7 cells identified a set of genes involved in osteoclastogenesis potentially regulated by Zfp687, e.g., Tspan7, Cpe, Vegfc, and Ggt1, confirming its role in this process. Strikingly, in this mouse model, the mutation was also associated with a high penetrance of hepatocellular carcinomas. Thus, this study established an essential role of Zfp687 in the regulation of bone remodeling, offering the potential to therapeutically treat PDB, and underlines the oncogenic potential of ZNF687.
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Pheochromocytoma/paragangliomas (PPGLs) are neuroendocrine tumours, often non-metastatic, but without available effective treatment for their metastatic form. Recent studies have shown that metformin exhibits antiproliferative activity in many human cancers, including PPGLs. Nevertheless, no data are available on the role of metformin on PPGL cells (two-dimension, 2D) and spheroids (three-dimension, 3D) migration/invasion. In this study, we observed that metformin exerts an antiproliferative effect on 2D and 3D cultures of pheochromocytoma mouse tumour tissue (MTT), either silenced or not for the SDHB subunit. However, metformin did not affect MTT migration. On the other hand, metformin did not have a short-term effect on the proliferation of mouse primary fibroblasts, but significantly decreased their ability to migrate. Although the metabolic changes induced by metformin were similar between MTT and fibroblasts (i.e., an overall decrease of ATP production and an increase in intracellular lactate concentration) the activated signalling pathways were different. Indeed, after metformin administration, MTT showed a reduced phosphorylation of Akt and Erk1/2, while fibroblasts exhibited a downregulation of N-Cadherin and an upregulation of E-Cadherin. Herein, we demonstrated that metformin has different effects on cell growth and spread depending on the cell type nature, underlining the importance of the tumour microenvironment in dictating the drug response.
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The expression of the nuclear receptor transcription factor (TF) COUPTFII is broadly associated with cell differentiation and cancer development, including of pancreatic ductal adenocarcinoma (PDAC), a devastating disease with one of the poorest prognoses among cancers worldwide. Recent studies have started to investigate the pathological and physiological roles of a novel COUPTFII isoform (COUPTFII_V2) that lacks the DNAbinding domain. As the role of the canonical COUPTFII in PDAC was previously demonstrated, the present study evaluated whether COUPTFII_V2 may have a functional role in PDAC. It was demonstrated that COUPTFII_V2 naturally occurs in PDAC cells and in primary samples, where its expression is consistent with shorter overall survival and peripheral invasion. Of note, COUPTFII_V2, exhibiting nuclear and cytosolic expression, is linked to epithelial to mesenchymal transition (EMT) and cancer progression, as confirmed by nude mouse experiments. The present results demonstrated that COUPTFII_V2 distinctively regulates the EMT of PDAC and, similarly to its sibling, it is associated with tumor aggressiveness. The two isoforms have both overlapping and exclusive functions that cooperate with cancer growth and dissemination. By studying how PDAC cells switch from one isoform to the other, novel insight into cancer biology was gained, indicating that this receptor may serve as a novel possible target for PDAC management.
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Fator II de Transcrição COUP/genética , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/genética , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Receptores Nucleares Órfãos , Neoplasias Pancreáticas/genética , Isoformas de Proteínas/genéticaRESUMO
Germline mutations in more than 20 genes, including those encoding for the succinate dehydrogenase (SDH), predispose to rare tumours, such as pheochromocytoma/paraganglioma (PPGL). Despite encoding for the same enzymatic complex, SDHC and SDHD mutated PHEO/PGLs are generally benign, while up to 80% of SDHB mutated ones are malignant. In this study, we evaluated the different effects of tumour microenvironment on tumour cell migration/invasion, by co-culturing SDHB or SDHD silenced tumour spheroids with primary cancer-associated fibroblasts (CAFs). We observed that SDHD silenced spheroids had an intermediate migration pattern, compared to the highest migration capability of SDHB and the lowest one of the wild type (Wt) spheroids. Interestingly, we noticed that co-culturing Wt, SDHB and SDHD silenced spheroids with CAFs in low glucose (1 g/l) medium, caused a decreased migration of all the spheroids, but only for SDHB silenced ones this reduction was significant. Moreover, the collective migration, observed in high glucose (4.5 g/l) and characteristic of the SDHB silenced cells, was completely lost in low glucose. Importantly, migration could not be recovered even adding glucose (3.5 g/l) to low glucose conditioned medium. When we investigated cell metabolism, we found that low glucose concentration led to a reduction of oxygen consumption rate (OCR), basal and maximal oxidative metabolism, and ATP production only in CAFs, but not in tumour cells. These results suggest that CAFs metabolism impairment was responsible for the decreased invasion process of tumour cells, most likely preventing the release of the pro-migratory factors produced by CAFs. In conclusion, the interplay between CAFs and tumour cells is distinctive depending on the gene involved, and highlights the possibility to inhibit CAF-induced migration by impairing CAFs metabolism, indicating new potential therapeutic scenarios for medical therapy.
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Neoplasias das Glândulas Suprarrenais , Paraganglioma , Feocromocitoma , Neoplasias das Glândulas Suprarrenais/metabolismo , Mutação em Linhagem Germinativa , Humanos , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Microambiente TumoralRESUMO
The bioimaging of cancer cells by the specific targeting of overexpressed biomarkers is an approach that holds great promise in the identification of selective diagnostic tools. Tumor-associated human carbonic anhydrase (hCA) isoforms IX and XII have been considered so far as well-defined biomarkers, with their expression correlating with cancer progression and aggressiveness. Therefore, the availability of highly performant fluorescent tools tailored for their targeting and able to efficiently visualize such key targets is in high demand. We report here on the design and synthesis of a kind of quantum dot (QD)-based fluorescent glyconanoprobe coated with a binary mixture of ligands, which, according to the structure of the terminal domains, impart specific property sets to the fluorescent probe. Specifically, monosaccharide residues ensured the dispersibility in the biological medium, CA inhibitor residues provided specific targeting of membrane-anchored hCA IX overexpressed on bladder cancer cells, and the quantum dots imparted the optical/fluorescence properties.
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BACKGROUND: Neuropathic pain is a clinically relevant adverse effect of several anticancer drugs that markedly impairs patients' quality of life and frequently leads to dose reduction or therapy discontinuation. The poor knowledge about the mechanisms involved in neuropathy development and pain chronicization, and the lack of effective therapies, make treatment of chemotherapy-induced neuropathic pain an unmet medical need. In this context, the vascular endothelial growth factor A (VEGF-A) has emerged as a candidate neuropathy hallmark and its decrease has been related to pain relief. In the present study, we have investigated the role of VEGF-A and its receptors, VEGFR-1 and VEGFR-2, in pain signalling and in chemotherapy-induced neuropathy establishment as well as the therapeutic potential of receptor blockade in the management of pain. METHODS: Behavioural and electrophysiological analyses were performed in an in vivo murine model, by using selective receptor agonists, blocking monoclonal antibodies or siRNA-mediated silencing of VEGF-A and VEGFRs. Expression of VEGF-A and VEGFR-1 in astrocytes and neurons was detected by immunofluorescence staining and confocal microscopy analysis. RESULTS: In mice, the intrathecal infusion of VEGF-A (VEGF165 isoforms) induced a dose-dependent noxious hypersensitivity and this effect was mediated by VEGFR-1. Consistently, electrophysiological studies indicated that VEGF-A strongly stimulated the spinal nociceptive neurons activity through VEGFR-1. In the dorsal horn of the spinal cord of animals affected by oxaliplatin-induced neuropathy, VEGF-A expression was increased in astrocytes while VEGFR-1 was mainly detected in neurons, suggesting a VEGF-A/VEGFR-1-mediated astrocyte-neuron cross-talk in neuropathic pain pathophysiology. Accordingly, the selective knockdown of astrocytic VEGF-A by intraspinal injection of shRNAmir blocked the development of oxaliplatin-induced neuropathic hyperalgesia and allodynia. Interestingly, both intrathecal and systemic administration of the novel anti-VEGFR-1 monoclonal antibody D16F7, endowed with anti-angiogenic and antitumor properties, reverted oxaliplatin-induced neuropathic pain. Besides, D16F7 effectively relieved hypersensitivity induced by other neurotoxic chemotherapeutic agents, such as paclitaxel and vincristine. CONCLUSIONS: These data strongly support the role of the VEGF-A/VEGFR-1 system in mediating chemotherapy-induced neuropathic pain at the central nervous system level. Thus, treatment with the anti-VEGFR-1 mAb D16F7, besides exerting antitumor activity, might result in the additional advantage of attenuating neuropathic pain when combined with neurotoxic anticancer agents.
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Anticorpos Monoclonais/uso terapêutico , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Humanos , Masculino , Camundongos , Transdução de SinaisRESUMO
Hybrid materials composed of superparamagnetic iron oxide nanoparticles (SPIONs) and lipid self-assemblies possess considerable applicative potential in the biomedical field, specifically, for drug/nutrient delivery. Recently, we showed that SPIONs-doped lipid cubic liquid crystals undergo a cubic-to-hexagonal phase transition under the action of temperature or of an alternating magnetic field (AMF). This transition triggers the release of drugs embedded in the lipid scaffold or in the water channels. In this contribution, we address this phenomenon in depth, to fully elucidate the structural details and optimize the design of hybrid multifunctional carriers for drug delivery. Combining small-angle X-ray scattering (SAXS) with a magnetic characterization, we find that, in bulk lipid cubic phases, the cubic-to-hexagonal transition determines the magnetic response of SPIONs. We then extend the investigation from bulk liquid-crystalline phases to colloidal dispersions, i.e., to lipid/SPIONs nanoparticles with cubic internal structure ("magnetocubosomes"). Through Synchrotron SAXS, we monitor the structural response of magnetocubosomes while exposed to an AMF: the magnetic energy, converted into heat by SPIONs, activates the cubic-to-hexagonal transition, and can thus be used as a remote stimulus to spike drug release "on-demand". In addition, we show that the AMF-induced phase transition in magnetocubosomes steers the realignment of SPIONs into linear string assemblies and connect this effect with the change in their magnetic properties, observed at the bulk level. Finally, we assess the internalization ability and cytotoxicity of magnetocubosomes in vitro on HT29 adenocarcinoma cancer cells, in order to test the applicability of these smart carriers in drug delivery applications.
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Sistemas de Liberação de Medicamentos , Nanopartículas Magnéticas de Óxido de Ferro/química , Adenocarcinoma/tratamento farmacológico , Antineoplásicos/administração & dosagem , Liberação Controlada de Fármacos , Células HT29 , Humanos , Transição de Fase , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Cancer-associated fibroblasts (CAFs) are one of the main components of the stromal compartment in the tumor microenvironment (TME) and the crosstalk between CAFs and cancer cells is essential for tumor progression and aggressiveness. Cancer cells mediate an activation process, converting normal fibroblasts into CAFs, that are characterized by modified expression of many proteins and increased production and release of microvesicles (MVs), extracellular vesicles generated by outwards budding from the cell membrane. Recent evidence underlined that the uptake of CAF-derived MVs changes the overall protein content of tumor cells. In this paper, we demonstrate that tumor activated fibroblasts overexpress Galectin-1 (Gal-1) and consequently release MVs containing increased levels of this protein. The uptake of Gal-1 enriched MVs by tumor cells leads to the upregulation of its intracellular concentration, that strongly affects cancer cell migration, while neither proliferation nor adhesion are altered. Accordingly, tumor cells co-cultured with fibroblasts silenced for Gal-1 have a reduced migratory ability. The present work reveals the key role of an exogenous protein, Gal-1, derived from activated fibroblasts, in cancer progression, and contributes to clarify the importance of MVs-mediated protein trafficking in regulating tumor-stroma crosstalk.
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Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies. SIGNIFICANCE: This study uncovers a link between a central effector of DNA damage response, CHK2, and cellular metabolism, revealing potential therapeutic strategies for targeting hepatocellular carcinoma.
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Carcinoma Hepatocelular/patologia , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Glicólise , Neoplasias Hepáticas/patologia , Metaboloma , Transcriptoma , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Quinase do Ponto de Checagem 2/genética , Ciclo do Ácido Cítrico , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitose , Espécies Reativas de Oxigênio/metabolismo , Succinatos/metabolismoRESUMO
BACKGROUND: Capsule endoscopy has been considered the first-line approach for the investigation of obscure gastro-intestinal bleeding since its approval in 2001. Our study aims to evaluate the diagnostic yield of capsule endoscopy in the investigation of this condition. We also analyse the incidence of non-small-bowel lesions missed after conventional endoscopy and later detected by capsule endoscopy in patients with suspected obscure bleeding. METHODS: A total of 290 patients with negative conventional endoscopy referred to our centre to undergo a capsule endoscopy examination for the investigation of obscure gastro-intestinal bleeding. We considered as non-small-bowel lesions those outside the tract between the second duodenal portion and the ileocecal valve. We also looked for actively bleeding lesions at the time of the exam. RESULTS: Intestinal preparation was good, adequate or poor in 74.1%, 8.4%, and 17.5% of the tests, respectively. Caecum was reached in 92.4%. Capsule retention occurred in 0.7%. Mean small bowel transit time was 5hours and 13minutes. Diagnostic yield was 73.8%. An actively bleeding lesion was noticed in 39.3% of positive tests. Capsule endoscopy revealed clinically significant non-small-bowel lesions missed at gastroscopy or colonoscopy in 30.3% of patients, 43.2% of which were bleeding. CONCLUSIONS: Capsule endoscopy has high diagnostic yield and safety in the investigation of obscure gastro-intestinal bleedings. Given the high percentage of non-small-bowel lesions detected, it may be appropriate to consider an endoscopic second look before performing a capsule endoscopy study.
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Endoscopia por Cápsula , Colonoscopia , Endoscopia Gastrointestinal , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/etiologia , Gastroscopia , Humanos , Estudos RetrospectivosRESUMO
AIMS: Obesity represents a global health problem. Excessive caloric intake promotes the release of inflammatory mediators by hypertrophic adipocytes and obesity-induced inflammation is now recognized as a risk factor for the development of several diseases, such as cardiovascular diseases, insulin resistance, type-II diabetes, liver steatosis and cancer. Since obesity causes inflammation, we tested the ability of acetylsalicylic acid (ASA), a potent anti-inflammatory drug, in counteracting this inflammatory process and in mitigating obesity-associated health complications. MAIN METHODS: Mice were fed with standard (SD) or high fat diet (HFD) for 3 months and then treated with acetylsalicylic acid for the subsequent two months. We then analyzed the metabolic and inflammatory status of their adipose and liver tissue by histological, molecular and biochemical analysis. KEY FINDINGS: Although ASA did not exert any effect on body weight, quantification of adipocyte size revealed that the drug slightly reduced adipocyte hypertrophy, however not sufficient so as to induce weight loss. Most importantly, ASA was able to improve insulin resistance. Gene expression profiles of pro- and anti-inflammatory cytokines as well as the expression of macrophage and lymphocyte markers revealed that HFD led to a marked macrophage accumulation in the adipose tissue and an increase of several pro-inflammatory cytokines, a situation almost completely reverted after ASA administration. In addition, liver steatosis caused by HFD was completely abrogated by ASA treatment. SIGNIFICANCE: ASA can efficiently ameliorate pathological conditions usually associated with obesity by inhibiting the inflammatory process occurring in the adipose tissue.
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Tecido Adiposo/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/uso terapêutico , Aspirina/uso terapêutico , Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina/fisiologia , Obesidade/tratamento farmacológico , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Modelos Animais de Doenças , Feminino , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Obesidade/patologia , Resultado do TratamentoRESUMO
Humans are continually exposed to a large number of environmental carcinogens [...].
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Dano ao DNA , Monitoramento Ambiental , Exposição Ocupacional , Estresse Oxidativo , Doença Crônica , HumanosRESUMO
Azathioprine is a cornerstone of the therapy of Crohn's disease. Unfortunately, infections and malignancies are relatively common adverse effects related to this drug; however, cirrhosis is exceptionally reported as a side effect. We report the case of a 49-year-old male patient with ileocolonic steno-penetrating Crohn's disease who developed hepatic cirrhosis while treated with azathioprine. After taking azathioprine for 3 years with regular follow-up, he developed pancytopenia, and liver cirrhosis was diagnosed with ultrasound, abdomen computed tomography scan, transient elastography, and liver biopsy. As all other causes of liver damage were excluded, azathioprine was believed to be the cause of liver injury and therefore was interrupted.
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It has been well established, particularly in animal models, that oestrogens exert neuroprotective effects in brain areas linked to cognitive processes. A key protective role could reside in the capacity of oestrogen to modulate the inflammatory response. However, the direct neuroprotective actions of oestrogens on neurones are complex and remain to be fully clarified. In the present study, we took advantage of a previously characterised primary culture of human cholinergic neurones (hfNBM) from the foetal nucleus basalis of Meynert, which is known to regulate hippocampal and neocortical learning and memory circuits, aiming to investigate the direct effects of oestrogens under inflammatory conditions. Exposure of cells to tumour necrosis factor (TNF)α (10 ng mL-1 ) determined the activation of an inflammatory response, as demonstrated by nuclear factor-kappa B p65 nuclear translocation and cyclooxygenase-2 mRNA expression. These effects were inhibited by treatment with either 17ß-oestradiol (E2 ) (10 nmol L-1 ) or G1 (100 nmol L-1 ), the selective agonist of the G protein-coupled oestrogen receptor (GPER1). Interestingly, the GPER1 antagonist G15 abolished the effects of E2 in TNFα-treated cells, whereas the ERα/ERß inhibitor tamoxifen did not. Electrophysiological measurements in hfNBMs revealed a depolarising effect caused by E2 that was specifically blocked by tamoxifen and not by G15. Conversely, G1 specifically hyperpolarised the cell membrane and also increased both inward and outward currents elicited by a depolarising stimulus, suggesting a modulatory action on hfNBM excitability by GPER1 activation. Interestingly, pretreating cells with TNFα completely blocked the effects of G1 on membrane properties and also significantly reduced GPER1 mRNA expression. In addition, we found a peculiar subcellular localisation of GPER1 to focal adhesion sites that implicates new possible mechanisms of action of GPER1 in the neuronal perception of mechanical stimuli. The results obtained in the present study indicate a modulatory functional role of GPER1 with respect to mediating the oestrogen neuroprotective effect against inflammation in brain cholinergic neurones and, accordingly, may help to identify protective strategies for preventing cognitive impairments.
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Anti-Inflamatórios/farmacologia , Núcleo Basal de Meynert/efeitos dos fármacos , Neurônios Colinérgicos/efeitos dos fármacos , Ciclopentanos/farmacologia , Estradiol/farmacologia , Estrogênios/farmacologia , Quinolinas/farmacologia , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Núcleo Basal de Meynert/metabolismo , Neurônios Colinérgicos/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfaRESUMO
RuvBL1 is an AAA+ ATPase whose expression in hepatocellular carcinoma (HCC) correlates with a poor prognosis. In vitro models suggest that targeting RuvBL1 could be an effective strategy against HCC. However, the role of RuvBL1 in the onset and progression of HCC remains unknown. To address this question, we developed a RuvBL1hep+/- mouse model and evaluated the outcome of DEN-induced liver carcinogenesis up to 12 months of progression. We found that RuvBL1 haploinsufficiency initially delayed the onset of liver cancer, due to a reduced hepatocyte turnover in RuvBL1hep+/- mice. However, RuvBL1hep+/- mice eventually developed HCC nodules that, with aging, grew larger than in the control mice. Moreover, RuvBL1hep+/- mice developed hepatic insulin resistance and impaired glucose homeostasis. We could determine that RuvBL1 regulates insulin signaling through the Akt/mTOR pathway in liver physiology in vivo as well as in normal hepatocytic and HCC cells in vitro. Whole transcriptome analysis of mice livers confirmed the major role of RuvBL1 in the regulation of hepatic glucose metabolism. Finally, RuvBL1 expression was found significantly correlated to glucose metabolism and mTOR signaling by bioinformatic analysis of human HCC sample from the publicly available TGCA database. These data uncover a role of RuvBL1 at the intersection of liver metabolism, hepatocyte proliferation and HCC development, providing a molecular rationale for its overexpression in liver cancer.
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ATPases Associadas a Diversas Atividades Celulares/genética , Carcinoma Hepatocelular/genética , Proteínas de Transporte/genética , DNA Helicases/genética , Resistência à Insulina/genética , Neoplasias Hepáticas/genética , Fígado/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Animais , Carcinogênese/genética , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Estudos de Coortes , DNA Helicases/metabolismo , Conjuntos de Dados como Assunto , Dietilnitrosamina/administração & dosagem , Dietilnitrosamina/toxicidade , Modelos Animais de Doenças , Progressão da Doença , Intervalo Livre de Doença , Glucose/metabolismo , Haploinsuficiência , Hepatócitos/metabolismo , Humanos , Insulina/metabolismo , Fígado/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Regulação para CimaRESUMO
Cadmium (Cd) is a highly toxic environmental pollutant released from the smelting and refining of metals and cigarette smoking. Oral exposure to cadmium may result in adverse effects on a number of tissues, including the central nervous system (CNS). In fact, its toxicity has been related to neurological disorders, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Under normal conditions, Cd barely reaches the brain in adults because of the presence of the blood-brain barrier (BBB); however, it has been demonstrated that Cd-dependent BBB alteration contributes to pathogenesis of neurodegeneration. However, the mechanism underlying Cd-dependent BBB alteration remain obscure. Here, we investigated the signaling pathway of Cd-induced tight junction (TJ), F-actin, and vimentin protein disassembly in a rat brain endothelial cell line (RBE4). RBE4 cells treated with 10 µM cadmium chloride (CdCl2) showed a dose- and time-dependent significant increase in reactive oxygen species (ROS) production. This phenomenon was coincident with the alteration of the TJ zonula occludens-1 (ZO-1), F-actin, and vimentin proteins. The Cd-dependent ROS increase elicited the upregulation of GRP78 expression levels, a chaperone involved in endoplasmic reticulum (ER) stress that induces caspase-3 activation. Further signal profiling by the pannexin-1 (PANX1) specific inhibitor 10Panx revealed a PANX1-independent increase in ATP spillage in Cd-treated endothelial cells. Our results point out that a ROS-dependent ER stress-mediated signaling pathway involving caspase-3 activation and ATP release is behind the BBB morphological alterations induced by Cd.
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Barreira Hematoencefálica/metabolismo , Cádmio/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Actinas/metabolismo , Animais , Barreira Hematoencefálica/citologia , Linhagem Celular , Estresse do Retículo Endoplasmático , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vimentina/metabolismoRESUMO
As the main metabolic and detoxification organ, the liver constantly adapts its activity to fulfill the energy requirements of the whole body. Despite the remarkable adaptive capacity of the liver, prolonged exposure to noxious stimuli such as alcohol, viruses and metabolic disorders results in the development of chronic liver disease that can progress to hepatocellular carcinoma (HCC), which is currently the second leading cause of cancer-related death worldwide. Metabolic rewiring is a common feature of cancers, including HCC. Altered mito-nuclear communication is emerging as a driving force in the metabolic reprogramming of cancer cells, affecting all aspects of cancer biology from neoplastic transformation to acquired drug resistance. Here, we explore relevant aspects (and discuss recent findings) of mito-nuclear crosstalk in the metabolic reprogramming of hepatocellular carcinoma.
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Carcinoma Hepatocelular/metabolismo , Núcleo Celular/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Animais , Carcinoma Hepatocelular/genética , Epigênese Genética , Humanos , Neoplasias Hepáticas/genética , Transdução de SinaisRESUMO
OBJECTIVE: Chromosomal instability (CIN) is the most common form of genomic instability, which promotes hepatocellular carcinoma (HCC) progression by enhancing tumour heterogeneity, drug resistance and immunity escape. CIN per se is an important factor of DNA damage, sustaining structural chromosome abnormalities but the underlying mechanisms are unknown. DESIGN: DNA damage response protein checkpoint kinase 2 (Chk2) expression was evaluated in an animal model of diethylnitrosamine-induced HCC characterised by DNA damage and elevated mitotic errors. Chk2 was also determined in two discrete cohorts of human HCC specimens. To assess the functional role of Chk2, gain on and loss-of-function, mutagenesis, karyotyping and immunofluorescence/live imaging were performed by using HCT116, Huh7 and human hepatocytes immortalised with hTERT gene (HuS). RESULTS: We demonstrate that mitotic errors during HCC tumorigenesis cause lagging chromosomes/DNA damage and activation of Chk2. Overexpression/phosphorylation and mislocalisation within the mitotic spindle of Chk2 contributes to induce lagging chromosomes. Lagging chromosomes and mitotic activity are reversed by knockdown of Chk2. Furthermore, upregulated Chk2 maintains mitotic activity interacting with Aurora B kinase for chromosome condensation and cytokinesis. The forkhead-associated domain of Chk2 is required for Chk2 mislocalisation to mitotic structures. In addition, retinoblastoma protein phosphorylation contributes to defective mitoses. A cohort and independent validation cohort show a strong cytoplasm to nuclear Chk2 translocation in a subset of patients with HCC. CONCLUSIONS: The study reveals a new mechanistic insight in the coinvolvement of Chk2 in HCC progression. These findings propose Chk2 as a putative biomarker to detect CIN in HCC providing a valuable support for clinical/therapeutical management of patients.