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The aims of our study are to: (i) investigate the ability of nicotine to modulate the expression level of inflammatory cytokines in A549 cells infected with SARS-CoV-2; (ii) elucidate the ultrastructural features caused by the combination nicotine+SARS-CoV-2; and (iii) demonstrate the mechanism of action. In this study, A549 cells pretreated with nicotine were either exposed to LPS or poly(I:C), or infected with SARS-CoV-2. Treated and untreated cells were analyzed for cytokine production, cytotoxicity, and ultrastructural modifications. Vero E6 cells were used as a positive reference. Cells pretreated with nicotine showed a decrease of IL6 and TNFα in A549 cells induced by LPS or poly(I:C). In contrast, cells exposed to SARS-CoV-2 showed a high increase of IL6, IL8, IL10 and TNFα, high cytopathic effects that were dose- and time-dependent, and profound ultrastructural modifications. These modifications were characterized by membrane ruptures and fragmentation, the swelling of cytosol and mitochondria, the release of cytoplasmic content in extracellular spaces (including osmiophilic granules), the fragmentation of endoplasmic reticulum, and chromatin disorganization. Nicotine increased SARS-CoV-2 cytopathic effects, elevating the levels of inflammatory cytokines, and inducing severe cellular damage, with features resembling pyroptosis and necroptosis. The protective role of nicotine in COVID-19 is definitively ruled out.
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Nicotina , SARS-CoV-2 , Células A549 , COVID-19 , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Interleucina-6 , Lipopolissacarídeos , Nicotina/efeitos adversos , Nicotina/farmacologia , Fator de Necrose Tumoral alfaRESUMO
Tumor hypoxic microenvironment causes hypoxia inducible factor 1 alpha (HIF-1α) activation and necrosis with alarmins release. Importantly, HIF-1α also controls the expression of alarmin receptors in tumor cells that can bind to and be activated by alarmins. Human tumor tissues possess 1-2% of cancer stem cells (CSCs) residing in hypoxic niches and responsible for the metastatic potential of tumors. Our hypothesis is that hypoxic CSCs express alarmin receptors that can bind alarmins released during necrosis, an event favoring CSCs migration. To investigate this aspect, glioblastoma stem-like cell (GSC) lines were kept under hypoxia to determine the expression of hypoxic markers as well as receptor for advanced glycation end products (RAGE). The presence of necrotic extracts increased migration, invasion and cellular adhesion. Importantly, HIF-1α inhibition by digoxin or acriflavine prevented the response of GSCs to hypoxia alone or plus necrotic extracts. In vivo, GSCs injected in one brain hemisphere of NOD/SCID mice were induced to migrate to the other one in which a necrotic extract was previously injected. In conclusion, our results show that hypoxia is important not only for GSCs maintenance but also for guiding their response to external necrosis. Inhibition of hypoxic pathway may therefore represent a target for preventing brain invasion by glioblastoma stem cells (GSCs).
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Glioblastoma/etiologia , Glioblastoma/metabolismo , Hipóxia/metabolismo , Necrose/patologia , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral , Animais , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Microambiente Tumoral/genéticaRESUMO
Cardiomyopathies are myocardial disorders in which heart muscle is structurally and/or functionally abnormal. Previously, structural cardiomyocyte disorders due to adrenal diseases, such as hyperaldosteronism, hypercortisolism, and hypercatecholaminism, were misunderstood, and endomyocardial biopsy (EMB) was not performed because was considered dangerous and too invasive. Recent data confirm that, if performed in experienced centers, EMB is a safe technique and gives precious information about physiopathological processes implied in clinical abnormalities in patients with different systemic disturbances. In this review, we illustrate the most important features in patients affected by primary aldosteronism (PA), Cushing's syndrome (CS), and pheochromocytoma (PHEO). Then, we critically describe microscopic and ultrastructural aspects that have emerged from the newest EMB studies. In PA, the autonomous hypersecretion of aldosterone induces the alteration of ion and water homeostasis, intracellular vacuolization, and swelling; interstitial oedema could be a peculiar feature of myocardial toxicity. In CS, cardiomyocyte hypertrophy and myofibrillolysis could be related to higher expression of atrogin-1. Finally, in PHEO, the hypercontraction of myofilaments with the formation of contraction bands and occasional cellular necrosis has been observed. We expect to clear the role of EMB in patients with cardiomyopathies and adrenal disease, and we believe EMB is a valid tool to implement new management and therapies.
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Doenças das Glândulas Suprarrenais/complicações , Doenças das Glândulas Suprarrenais/patologia , Cardiomiopatias/etiologia , Cardiomiopatias/patologia , Doenças das Glândulas Suprarrenais/diagnóstico , Doenças das Glândulas Suprarrenais/metabolismo , Aldosterona/metabolismo , Animais , Biópsia , Cardiomiopatias/diagnóstico , Cardiomiopatias/metabolismo , Catecolaminas/metabolismo , Endocárdio/metabolismo , Endocárdio/patologia , Humanos , Hidrocortisona/metabolismo , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
BACKGROUND: Chronic rhinitis, pharyngitis and sinusitis are common health problems with a significant impact on public health, and are suspected to be influenced by ageing factors. Nasal inhalation with thermal water may be used to reduce symptoms, inflammation and drug intake. A pre-post clinical study was conducted in 183 consecutive adult and elderly patients with chronic rhinitis, pharyngitis or sinusitis, to evaluate whether thermal water nasal inhalations could improve their symptoms, clinical signs and rhinomanometry measurements, and influence inflammatory biomarkers levels in nasal epithelial cells. RESULTS: Participants profile revealed that they were aged on average (mean age and SD 60.6 ± 15.2 years, median 65, range 20-86, 86 aged ≤ 65 years (47%), 96 aged > 65 years (53%)) and extremely concerned about wellbeing. Older age was associated with better compliance to inhalation treatment. Total symptom and clinical evaluation scores were significantly ameliorated after treatment (p < 0.001), with no substantial difference according to age, while rhinomanometry results were inconsistent. Persistence of symptom improvement was confirmed at phone follow up 1 year later (n = 74). The training set of 48 inflammatory genes (40 patients) revealed a strong increase of CXCR4 gene expression after nasal inhalations, confirmed both in the validation set (143 patients; 1.2 ± 0.68 vs 3.3 ± 1.2; p < 0.0001) and by evaluation of CXCR4 protein expression (40 patients; 1.0 ± 0.39 vs 2.6 ± 0.66; p < 0.0001). CXCR4 expression was consistently changed in patients with rhinitis, pharyngitis or sinusitis. The increase was smaller in current smokers compared to non-smokers. Results were substantially unchanged when comparing aged subjects (≥ 65 years) or the eldest quartile (≥ 71 years) to the others. Other genes showed weaker variations (e.g. FLT1 was reduced only in patients with sinusitis). CONCLUSIONS: These results confirm the clinical impact of thermal water nasal inhalations on upper respiratory diseases both in adults and elders, and emphasize the role of genes activating tissue repair and inflammatory pathways. Future studies should evaluate CXCR4 as possible therapeutic target or response predictor in patients with chronic rhinitis, pharyngitis or sinusitis. TRIAL REGISTRATION: Communication to Italian Ministry of Health - ICPOM 000461. Registered 10/11/2014.
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Exogenous High Mobility Group Box-1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex. In particular, its effect on ischemic cardiomyocytes has been poorly investigated. Aim of the present study was to verify whether and how autophagy and apoptosis were involved in HMGB1-induced heart repair following myocardial infarction (MI). HMGB1 (200 ng) or denatured HMGB1 were injected in the peri-infarcted region of mouse hearts following acute MI. Three days after treatment, an upregulation of autophagy was detected in infarcted HMGB1 treated hearts compared to controls. Specifically, HMGB1 induced autophagy by significantly upregulating the protein expression of LC3, Beclin-1, and Atg7 in the border zone. To gain further insights into the molecular mechanism of HMGB1-mediated autophagy, WB analysis were performed in cardiomyocytes isolated from 3 days infarcted hearts in the presence and in the absence of HMGB1 treatment. Results showed that upregulation of autophagy by HMGB1 treatment was potentially related to activation of AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Accordingly, in these hearts, phospho-Akt signaling pathway was inhibited. The induction of autophagy was accompanied by reduced cardiomyocyte apoptotic rate and decreased expression levels of Bax/Bcl-2 and active caspase-3 in the border zone of 3 days infarcted mice following HMGB1 treatment. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP-activated protein kinase-dependent autophagy. J. Cell. Physiol. 232: 1135-1143, 2017. © 2016 Wiley Periodicals, Inc.
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Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína HMGB1/farmacologia , Complexos Multiproteicos/antagonistas & inibidores , Infarto do Miocárdio/patologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Biomarcadores/metabolismo , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Testes de Função Cardíaca , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/metabolismo , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Sirtuins are conserved NAD+ -dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1-SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1-SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1-silenced cell lines. Our results demonstrate that SIRT1-silenced cells are more resistant to H2 O2 and etoposide treatment showing decreased ROS accumulation, γ-H2AX phosphorylation, caspase-3 activation and PARP cleavage. Interestingly, we observed that SIRT1-silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1-silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co-immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1-silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835-1844, 2017. © 2016 Wiley Periodicals, Inc.
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Etoposídeo/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/metabolismo , Sirtuína 3/metabolismo , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição Sp1/metabolismoRESUMO
Low-grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of two subpopulations of adipose-derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of two subpopulations of ASC from the same isolation sites of non-obese individuals. In particular, the behavior of ASC of obese versus non-obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV-ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation-related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue-specific NF-kB activation and an NF-kB-mediated increase in inflammatory and fibrogenic responses. Moreover, obV-ASC, which showed a less stem-like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV-ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF-kB inhibitors. These novel findings suggest that the deregulated hyper-responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low-grade chronic inflammation, which has been implicated in obesity-related morbidity.
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Adipócitos/citologia , Diferenciação Celular/fisiologia , Gordura Intra-Abdominal/citologia , Obesidade/metabolismo , Células-Tronco/citologia , Tecido Adiposo/metabolismo , Adulto , Idoso , Hipóxia Celular , Células Cultivadas , Feminino , Humanos , Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Gordura Subcutânea/citologiaRESUMO
Lymphatic dissemination from the primary tumor is a major mechanism by which breast cancer cells access the systemic circulation, resulting in distant metastasis and mortality. Numerous studies link activation of hypoxia-inducible factor 1 (HIF-1) with tumor angiogenesis, metastasis, and patient mortality. However, the role of HIF-1 in lymphatic dissemination is poorly understood. In this study, we show that HIF-1 promotes lymphatic metastasis of breast cancer by direct transactivation of the gene encoding platelet-derived growth factor B (PDGF-B), which has proliferative and chemotactic effects on lymphatic endothelial cells. Lymphangiogenesis and lymphatic metastasis in mice bearing human breast cancer orthografts were blocked by administration of the HIF-1 inhibitor digoxin or the tyrosine kinase inhibitor imatinib. Immunohistochemical analysis of human breast cancer biopsies demonstrated colocalization of HIF-1α and PDGF-B, which were correlated with lymphatic vessel area and histological grade. Taken together, these data provide experimental support for breast cancer clinical trials targeting HIF-1 and PDGF-B.
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Neoplasias da Mama/fisiopatologia , Fator 1 Induzível por Hipóxia/fisiologia , Metástase Linfática/fisiopatologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Ativação Transcricional/fisiologia , Benzamidas , Neoplasias da Mama/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Digoxina/farmacologia , Feminino , Humanos , Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Mesilato de Imatinib , Immunoblotting , Imuno-Histoquímica , Luciferases , Linfangiogênese/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
Growing evidence indicates that microRNAs (miRNAs or miRs) are involved in basic cell functions and oncogenesis. Here we report that miR-133 has a critical role in determining cardiomyocyte hypertrophy. We observed decreased expression of both miR-133 and miR-1, which belong to the same transcriptional unit, in mouse and human models of cardiac hypertrophy. In vitro overexpression of miR-133 or miR-1 inhibited cardiac hypertrophy. In contrast, suppression of miR-133 by 'decoy' sequences induced hypertrophy, which was more pronounced than that after stimulation with conventional inducers of hypertrophy. In vivo inhibition of miR-133 by a single infusion of an antagomir caused marked and sustained cardiac hypertrophy. We identified specific targets of miR-133: RhoA, a GDP-GTP exchange protein regulating cardiac hypertrophy; Cdc42, a signal transduction kinase implicated in hypertrophy; and Nelf-A/WHSC2, a nuclear factor involved in cardiogenesis. Our data show that miR-133, and possibly miR-1, are key regulators of cardiac hypertrophy, suggesting their therapeutic application in heart disease.
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Cardiomegalia/genética , MicroRNAs/genética , Animais , Aorta Torácica/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Oncogênica v-akt/genética , RatosRESUMO
[This corrects the article DOI: 10.3389/fendo.2024.1340188.].
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Introduction: Fabry's disease (FD) is a genetic X-linked systemic and progressive rare disease characterized by the accumulation of globotriaosylceramide (GB3) into the lysosomes of many tissues. FD is due to loss-of-function mutations of α-galactosidase, a key-enzyme for lysosomal catabolism of glycosphingolipids, which accumulate as glycolipid bodies (GB). In homozygous males the progressive deposition of GB3 into the cells leads to clinical symptoms in CNS, skin, kidney, etc. In testis GB accumulation causes infertility and alterations of spermatogenesis. However, the precise damaging mechanism is still unknown. Our hypothesis is that GB accumulation reduces blood vessel lumen and increases the distance of vessels from both stromal cells and seminiferous parenchyma; this, in turn, impairs oxygen and nutrients diffusion leading to subcellular degradation of seminiferous epithelium and sterility. Methods: To test this hypothesis, we have studied a 42-year-old patient presenting a severe FD and infertility, with reduced number of spermatozoa, but preserved sexual activity. Testicular biopsies were analyzed by optical (OM) and transmission electron microscopy (TEM). Activation and cellular localization of HIF-1α and NFκB was analyzed by immunofluorescence (IF) and RT-PCR on homogeneous tissue fractions after laser capture microdissection (LCMD). Results: OM and TEM showed that GB were abundant in vessel wall cells and in interstitial cells. By contrast, GB were absent in seminiferous epithelium, Sertoli's and Leydig's cells. However, seminiferous tubular epithelium and Sertoli's cells showed reduced diameter, thickening of basement membrane and tunica propria, and swollen or degenerated spermatogonia. IF showed an accumulation of HIF-1α in stromal cells but not in seminiferous tubules. On the contrary, NFκB fluorescence was evident in tubules, but very low in interstitial cells. Finally, RT-PCR analysis on LCMD fractions showed the expression of pro-inflammatory genes connected to the HIF-1α/NFκB inflammatory-like pathway. Conclusion: Our study demonstrates that infertility in FD may be caused by reduced oxygen and nutrients due to GB accumulation in blood vessels cells. Reduced oxygen and nutrients alter HIF-1α/NFκB expression and localization while activating HIF-1α/NFκB driven-inflammation-like response damaging seminiferous tubular epithelium and Sertoli's cells.
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Doença de Fabry , Infertilidade , Adulto , Humanos , Masculino , Doença de Fabry/complicações , Doença de Fabry/patologia , Hipóxia/patologia , Infertilidade/patologia , Inflamação/complicações , Inflamação/patologia , Oxigênio , Testículo/patologiaRESUMO
The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF-1 protein expression and secretion and of IGF-1R protein levels which, in turn, prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary, SIRT1 overexpression increased cell death. In particular, IGF-1 and IGF-1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF-1 and IGF-1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF-1 survival pathway.
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Fator de Crescimento Insulin-Like I/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Sobrevivência Celular , Regulação para Baixo/genética , Camundongos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , RNA Interferente Pequeno/genética , Ratos , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
Life on Earth has evolved in the presence of a gravity constraint. Any change in the value of such a constraint has important physiological effects. Gravity reduction (microgravity) alters the performance of muscle, bone and, immune systems among others. Therefore, countermeasures to limit such deleterious effects of microgravity are needed considering future Lunar and Martian missions. Our study aims to demonstrate that the activation of mitochondrial Sirtuin 3 (SIRT3) can be exploited to reduce muscle damage and to maintain muscle differentiation following microgravity exposure. To this effect, we used a RCCS machine to simulate microgravity on ground on a muscle and cardiac cell line. During microgravity, cells were treated with a newly synthesized SIRT3 activator, called MC2791 and vitality, differentiation, ROS and, autophagy/mitophagy were measured. Our results indicate that SIRT3 activation reduces microgravity-induced cell death while maintaining the expression of muscle cell differentiation markers. In conclusion, our study demonstrates that SIRT3 activation could represent a targeted molecular strategy to reduce muscle tissue damage caused by microgravity.
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Marte , Sirtuína 3 , Ausência de Peso , Meio Ambiente Extraterreno , Músculos/metabolismo , Sirtuína 3/metabolismo , HumanosRESUMO
The possible role of an exocytotic, vesicular mechanism in cellular volume regulation under iso-osmotic conditions has been studied in slices of rat liver. The effects of incubation conditions and agents affecting the actin cytoskeleton were examined for changes of water, ionic composition, and ultrastructure. Slices were pre-incubated at 1°C in an iso-osmotic buffered medium to induce swelling. Upon restoration to 37°C in the same medium, tissue lost water. The Na+-K+ adenosine triphosphatase (ATPase) inhibitor ouabain inhibited water extrusion of about 50%, an effect that was accompanied by the formation of characteristic vesicles in the cytoplasmic region between the Golgi apparatus and the bile canaliculi. Water extrusion in the presence of ouabain was partially inhibited by trifluoroperazine and completely inhibited when the medium was free of Ca2+. In the presence of ouabain, brefeldin A caused a small reduction of water extrusion, whereas phalloidin and cytochalasins A, D, or E caused a marked inhibition. In these conditions there was a marked increase in size and number of cytoplasmic vesicles and a more widespread distribution of them within the cells, lacking the more specific orientation to the Golgi and canalicular regions that was seen in the presence of ouabain alone. Water extrusion was inhibited by phalloidin and cytochalasins in the absence of ouabain. In conclusion, our results are consistent with the hypothesis that iso-osmotic expulsion of water from hepatocytes can proceed partly through an accumulation of water in cytoplasmic vesicles, followed by exocytosis. This mechanism does not depend on Na+-K+ ATPase activity.
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Cálcio/farmacologia , Forma Celular , Tamanho Celular , Hepatócitos/citologia , Hepatócitos/fisiologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Equilíbrio Hidroeletrolítico , Animais , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Cálcio/análise , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Citocalasinas/farmacologia , Citoesqueleto/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Técnicas In Vitro , Masculino , Ouabaína/farmacologia , Faloidina/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar , Trifluoperazina/farmacologia , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
Background: The efficacy of enzyme replacement therapy (ERT) in mobilizing globotryaosylceramide (GB-3) from Fabry cardiomyocytes is limited. The mechanism involved is still obscure. Methods: Assessment of M6Pr, M6Pr-mRNA, and Ubiquitin has been obtained by Western blot analysis and real-time PCR of frozen endomyocardial biopsy samples, from 17 pts with FD, various degree of left ventricular hypertrophy, and maximal wall thickening (MWT) from 11.5 and 20 mm. The diagnosis and severity of FDCM followed definitions of GLA mutation, α-galactosidase A enzyme activity, cardiac magnetic resonance, and left ventricular endomyocardial biopsy with the quantification of myocyte hypertrophy and the extent of Gb-3 accumulation. All patients have received alpha or beta agalsidase for ≥3 years without a reduction in LV mass nor an increase in T1 mapping at CMR. Controls were surgical biopsies from 15 patients undergoing mitral valve replacement. Results: Protein analysis showed mean M6Pr in FDCM to be 5.4-fold lower than in a normal heart (4289 ± 6595 vs. 23,581 ± 4074, p = 0.0996) (p < 0.001): specifically, 9-fold lower in males, p = 0.009, (p < 0.001) and 3-fold lower in females, p = 0.5799, (p < 0.001) showing, at histology, a mosaic of normal and diseased cells. M6Pr-mRNA expression was normal, while ubiquitin showed an increase of 4.6 fold vs. controls (13,284 ± 1723 vs. 2870 ± 690, p = 0.001) suggesting that ubiquitin-dependent post-translational degradation is likely responsible for the reduction of M6Pr in FDCM. Conclusion: M6Pr expression is remarkably reduced in FDCM as a likely result of post-translational degradation. This may explain the reduced efficacy of ERT and be a therapeutic target for the enhancement of ERT activity.
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Epithelial-mesenchymal transition (EMT), a physiological process during embryogenesis, can become pathological in the presence of different driving forces. Reduced oxygen tension or hypoxia is one of these forces, triggering a large number of molecular pathways with aberrant EMT induction, resulting in cancer and fibrosis onset. Both hypoxia-induced factors, HIF-1α and HIF-2α, act as master transcription factors implicated in EMT. On the other hand, hypoxia-dependent HIF-independent EMT has also been described. Recently, a new class of seven proteins with deacylase activity, called sirtuins, have been implicated in the control of both hypoxia responses, HIF-1α and HIF-2α activation, as well as EMT induction. Intriguingly, different sirtuins have different effects on hypoxia and EMT, acting as either activators or inhibitors, depending on the tissue and cell type. Interestingly, sirtuins and HIF can be activated or inhibited with natural or synthetic molecules. Moreover, recent studies have shown that these natural or synthetic molecules can be better conveyed using nanoparticles, representing a valid strategy for EMT modulation. The following review, by detailing the aspects listed above, summarizes the interplay between hypoxia, sirtuins, and EMT, as well as the possible strategies to modulate them by using a nanoparticle-based approach.
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The role of hypoxia in regulating tumor progression is still controversial. Here, we demonstrate that, similarly to what previously observed by us in human prostate and breast tumor samples, hypoxia increases expression of the receptor for advanced glycation end products (RAGE) and the purinergic receptor P2X7 (P2X7R). The role of hypoxia was shown by the fact that hypoxia-inducible factor (HIF)-1α silencing downregulated RAGE and P2X7R protein levels as well as nuclear factor-kappaB (NF-κB) expression. In contrast, NF-κB silencing reduced P2X7R expression without affecting RAGE protein levels or nuclear accumulation of HIF-1α. Treatment of hypoxic tumor cells with HMGB1 and BzATP ligands, respectively, of RAGE and P2X7R, activated a signaling pathway that, through Akt and Erk phosphorylation, determines nuclear accumulation of NF-κB and increases cell invasion. Inhibition of Akt by SH5 and Erk by INH1 prevented both nuclear translocation of NF-κB and cell invasion. Moreover, silencing RAGE and P2X7R abolished nuclear accumulation of NF-κB as well as cell invasion without affecting HIF-1α stabilization. Once in the nucleus, NF-κB would contribute to cell survival and invasion under hypoxia, by maintaining RAGE and P2X7R expression levels and matrix metalloproteinases 2 and 9 synthesis. These results show that, hypoxia can upregulate expression levels of membrane receptors that, by binding extracellular molecules eventually released by necrotic cells, contribute to the increased invasiveness of transformed tumor cells. Moreover, these observations strengthen our working hypothesis that upregulation of damage-associated molecular patterns receptors by HIF-1α represents the crucial event bridging hypoxia and inflammation in obtaining the malignant phenotype.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Imunológicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Immunoblotting , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/genética , Fosforilação , Transporte Proteico , RNA Interferente Pequeno/genética , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Transdução de SinaisRESUMO
The expression pattern of estrogen receptor (ER) isoforms in normal and tumor thyroid tissues is still controversial and poor defined, therefore, a more detailed study of the distribution of these molecules is needed. Most discrepancies might be due to the methods utilized. We studied the expression of ER isoforms in human papillary thyroid carcinoma (PTC), in fine-needle aspiration biopsy-derived specimens, and in cells, using more accurate techniques, such as laser-capture microdissection, real-time quantitative PCR, and Western blot. Laser-capture microdissection allowed us to isolate homogeneous cell populations from human PTC surgical samples. Tumor, peritumor, or normal host tissue of the same sample were separately dissected and analyzed by RT-PCR and Western blot. Estrogen receptor-α mRNA was more expressed in cancer-microdissected cells from human PTC, as compared with microdissected cells obtained from surrounding normal host tissue (450 vs 12, P = 0.001). A similar pattern was observed with Western blot for the ER-a protein. By contrast, ER-ß mRNA expression was not detected among the microdissected tissue fractions. Fine-needle aspiration biopsy-derived specimens showed a similar expression pattern to ER. Moreover, human PTC cell line BCPAP and cancer stem cells from PTC, analyzed under hypoxic conditions, showed a hypoxia-driven increase in ER-α expression. In conclusion, ER-α might have an important role in human PTC, and its overexpression can be studied in routine needle aspirate as a possible marker of malignancy.
Assuntos
Carcinoma Papilar/metabolismo , Receptor alfa de Estrogênio/biossíntese , Neoplasias da Glândula Tireoide/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Biópsia por Agulha Fina , Carcinoma , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia , Microdissecção e Captura a Laser , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
BACKGROUND: Adaptation to hypoxia and consequent pro-inflammatory gene expression of prostate and breast carcinomas have been implicated in the progression toward cancer malignant phenotype. Only partial data are available for the human tumor glioblastoma multiforme (GBM). The aim of our study was to analyze the hypoxic and pro-inflammatory microenvironment in GBMs and to demonstrate that in a stem/progenitor cell line derived from human glioblastoma (GBM-SCs), hypoxia activates a coordinated inflammatory response, evidencing an invasive and migratory phenotype. METHODS: From each of 10 human solid glioblastomas, clinically and histopathologically characterized, we obtained three surgical samples taken from the center and the periphery of the tumor, and from adjacent host normal tissue. Molecular and morphological analyses were carried out using quantitative real-time PCR and western blot (WB). GBM stem and differentiated cells were incubated under hypoxic conditions and analyzed for pro-inflammatory gene expression and for invasive/migratory behavior. RESULTS: A panel of selected representative pro-inflammatory genes (RAGE and P2X7R, COX2, NOS2 and, PTX3) were analyzed, comparing tumor, peritumor and host normal tissues. Tumors containing leukocyte infiltrates (as assessed using CD45 immunohistochemistry) were excluded. Selected genes were overexpressed in the central regions of the tumors (i.e. in the more hypoxic areas), less expressed in peripheral regions, and poorly expressed or absent in adjacent normal host tissues. Western blot analysis confirmed that the corresponding pro-inflammatory proteins were also differently expressed. Hypoxic stem cell lines showed a clear time-dependent activation of the entire panel of pro-inflammatory genes as compared to differentiated tumor cells. Biological assays showed that invasive and migratory behavior was strengthened by hypoxia only in GBM stem cells. CONCLUSIONS: In human solid glioblastoma we have observed a coordinated overexpression of a panel of pro-inflammatory genes as compared to host normal tissue. We have also evidenced a similar pattern of overexpressed genes in GBM-SCs after hypoxic treatment, showing also a gain of invasive and migratory function that was lost when these stem cells differentiated. We suggest that, as has been previously described for prostatic and mammary carcinoma, in human glioblastoma acquisition of a proinflammatory phenotype may be relevant for malignant progression.