RESUMO
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.
Assuntos
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
Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.
Assuntos
Autofagia/efeitos dos fármacos , Grafite/química , Grafite/farmacologia , Nanotubos de Carbono/química , Neuroblastoma/metabolismo , Óxidos/química , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Nanotubos de Carbono/ultraestrutura , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
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
Metformin has been shown to inhibit glutaminase (GLS) activity and ammonia accumulation thereby reducing the risk of hepatic encephalopathy in type 2 diabetic patients. Since tumour cells are addicted to glutamine and often show an overexpression of glutaminase, we hypothesize that the antitumoral mechanism of metformin could be ascribed to inhibition of GLS and reduction of ammonia and ammonia-induced autophagy. Our results show that, in different tumour cell lines, micromolar doses of metformin prevent cell growth by reducing glutamate, ammonia accumulation, autophagy markers such as MAP1LC3B-II and GABARAP as well as degradation of long-lived proteins. Reduced autophagy is then accompanied by increased BECN1/BCL2 binding and apoptotic cell death. Interestingly, GLS-silenced cells reproduce the effect of metformin treatment showing reduced MAP1LC3B-II and GABARAP as well as ammonia accumulation. Since metformin is used as adjuvant drug to increase the efficacy of Cisplatin-based neoadjuvant chemotherapy, we co-treated tumour cells with micromolar doses of metformin in the presence of cisplatin observing a marked reduction of MAP1LC3B-II and an increase of caspase 3 cleavage. In conclusion, our work demonstrates that the anti-tumoral action of metformin is due to the inhibition of glutaminase and autophagy and could be used to improve the efficacy of chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Glutaminase/antagonistas & inibidores , Glutamina/metabolismo , Metformina/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Amônia/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Cisplatino/farmacologia , Células HeLa , Humanos , Células MCF-7 , Proteínas Associadas aos Microtúbulos/metabolismo , Terapia NeoadjuvanteRESUMO
Coronary artery bypass grafting was performed in a 54-year-old man affected by untreated Fabry's disease. Left internal mammary artery (LIMA) and saphenous vein grafts were implanted. Surgical samples of LIMA revealed diffuse glycosphyngolipid infiltration of smooth muscle cells, whereas SV was normal. After surgery, the patient received antithrombotic and enzyme replacement therapy. At 1-year follow-up, LIMA graft occluded, whereas saphenous vein graft remained patent. In Fabry's disease, veins, probably because of a low pressure load, seem to be spared from glycosphingolipid accumulation and are more suitable than arteries for grafting. A preventive histology of conduits is suggested before graft selection.
Assuntos
Ponte de Artéria Coronária/métodos , Doença de Fabry/cirurgia , Oclusão de Enxerto Vascular/patologia , Artéria Torácica Interna/patologia , Artéria Torácica Interna/cirurgia , Veia Safena/patologia , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/cirurgia , Doença de Fabry/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Veia Safena/cirurgiaRESUMO
BACKGROUND: The objective of this study was to gain insight into the molecular mechanism of induced cell death (apoptosis) by PYRROLO [1,2-b][1,2,5]BENZOTHIADIAZEPINES (PBTDs) series compounds, using human (K562) cells as a model. METHODS: We focused our attention on some members of the PBTDs family to test their potential apoptotic activity in K562 cells. Important apoptotic activity was demonstrated, as evidenced by the concentration and percentage of cell death quantified by measuring PI-uptake by flow cytometry, and DNA fragmentation analyzed by agarose gel electrophoresis, generating a characteristic ladder pattern of discontinuous DNA fragments. The expression of Bcl-2 family was tested using western blotting and transfection method. RESULTS: PBTDs-mediated suppression of K562 cell proliferation was induced by apoptosis characterized by the appearance of DNA fragmentation and was associated with the poly(ADP-ribose)polymerase (PARP) cleavage. PBTD-1 and -3 treatment resulted in caspase-3 activation through down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we used K562/vector and K562/bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with K562/vector, K562/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 10 muM PBTD-1 and -3 for 24 h produced morphological features of apoptosis and DNA fragmentation in K562/vector cells, respectively. In contrast, PBTD-1 and -3-induced caspase-3 activation and apoptosis were inhibited in K562/Bcl-2. Furthermore, Bcl-2 overexpressing cells exhibited less cytocrome c release during PBTDs-induced apoptosis. CONCLUSION: These results indicate that PBTDs effectively induce apoptosis of K562 leukemia cells through the activation of caspase cascades. In addition, these findings indicate that Bcl-2 inhibits PBTD-1 and -3 induced-apoptosis via a mechanism that interferes with cytocrome c release, and the activity of caspase-3, which is involved in the execution of apoptosis.
Assuntos
Apoptose , Benzodiazepinas/farmacologia , Ciclo Celular/efeitos dos fármacos , Óxidos S-Cíclicos/farmacologia , Leucemia/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/efeitos dos fármacos , Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Regulação para Baixo , Genes bcl-2/fisiologia , Humanos , Células K562 , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-dioxides (PBTDs) induced apoptosis in human BCR-ABL-expressing leukemia cells. The apoptotic activity was also observed in primary leukemic blasts, obtained from chronic myelogenous leukemia (CML) patients at onset or from patients in blast crisis and who were imatinib-resistant. Compounds 5 and 14 induced apoptosis before BCR-ABL protein expression and tyrosin phosphorylation were affected and activated different caspases in the apoptotic pathway. PBTDs are a new class of valid candidates for the treatment of CML.
Assuntos
Antineoplásicos/síntese química , Apoptose , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Pirróis/síntese química , Tiazepinas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Proteínas de Fusão bcr-abl/biossíntese , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirróis/química , Pirróis/farmacologia , Tiazepinas/química , Tiazepinas/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Fabry disease (FD) has been recognized as the cause of left ventricular hypertrophy in 6% of men with late-onset hypertrophic cardiomyopathy (HCM). Although FD is considered a recessive X-linked disorder, affected women are increasingly reported. The aim of our study was to determine the prevalence of FD in female patients with HCM. METHODS AND RESULTS: Thirty-four consecutive women (mean age, 50+/-13.6 years) who received an ECG and echocardiographic diagnosis of HCM were submitted to an invasive cardiac study that included a biventricular endomyocardial biopsy. Tissue samples were analyzed for histology and electron microscopy. Peripheral blood activity of alpha-galactosidase (alpha-Gal) A was assessed in all patients. None of them had a family history of FD. Histology and electron microscopy showed in 4 patients (12%; mean age, 51.5+/-3.9 years) the presence of cell vacuoles characterized by the accumulation of glycolipid material organized in concentric lamellar structures, diagnostic for FD. In the remaining patients, histology was consistent with HCM. In all the female carriers, the heart was the only organ clinically involved in the disease, showing concentric hypertrophy in 2 patients, asymmetric hypertrophy in 1, and apical hypertrophy in 1. The alpha-Gal A enzymatic activity was 44+/-14% of control values. Genetic analysis showed the presence of alpha-Gal A gene mutation in all 4 cases. CONCLUSIONS: FD may account for up to 12% of females with late-onset HCM. Those heterozygous for FD with left ventricular hypertrophy are potential candidates for enzyme enhancement/replacement therapy.
Assuntos
Cardiomiopatia Hipertrófica/etiologia , Doença de Fabry/epidemiologia , Heterozigoto , Hipertrofia Ventricular Esquerda/etiologia , Adulto , Idade de Início , Idoso , Biópsia , Cardiomiopatia Hipertrófica/diagnóstico por imagem , Cardiomiopatia Hipertrófica/enzimologia , Cardiomiopatia Hipertrófica/epidemiologia , Análise Mutacional de DNA , Eletrocardiografia , Endocárdio/patologia , Doença de Fabry/complicações , Doença de Fabry/diagnóstico , Doença de Fabry/enzimologia , Doença de Fabry/genética , Feminino , Genótipo , Glicolipídeos/análise , Humanos , Hipertrofia Ventricular Esquerda/diagnóstico por imagem , Hipertrofia Ventricular Esquerda/epidemiologia , Lisossomos/enzimologia , Masculino , Pessoa de Meia-Idade , Linhagem , Prevalência , Ultrassonografia , Vacúolos/química , Vacúolos/ultraestrutura , alfa-Galactosidase/sangue , alfa-Galactosidase/genéticaRESUMO
AIMS: The pathogenesis of cocaine-related cardiomyopathy (CCM) is still unclear. Oxidative damage from cocaine-generated reactive oxygen species (ROS) overcoming myocardial antioxidant reserve has been hypothesized by experimental studies. METHODS AND RESULTS: Ten (2.3%) of 430 consecutive cases with dilated cardiomyopathy (DCM) were attributed to CCM. Endomyocardial biopsies from CCM were retrospectively investigated with histology, electron microscopy, immunohistochemistry (graded 0-3), and Western blot analysis for inducible nitric oxide synthase (iNOS) and nitrotyrosine. Oxidative damage to DNA was investigated by immunostaining for 8-hydroxydeoxyguanosine (8-OHdG), while apoptosis and necrosis were evaluated by in situ ligation with hairpin probes. Myocardial anti-oxidant reserve was evaluated through assessment of superoxide dismutase (SOD1-2) and catalase (CT) activity in two frozen samples from each patient. Results were compared with idiopathic DCM and normal controls. Cardiomyocytes were bigger and myocardial fibrosis was more pronounced in CCM than in the DCM cohort. Contraction band necrosis was always detectable only in CCM with sparse lymphocytic infiltrates in three cases. Both iNOS and nitrotyrosine were significantly more expressed in CCM than in DCM. Immunostaining for 8-OHdG, cardiomyocyte apoptosis, and necrosis were significantly increased in CCM compared with controls and DCM. Myocardial SOD1 and CT activity was significantly decreased compared with DCM and controls, and correlated with cell death and severity of left ventricular dysfunction. CONCLUSION: Oxidative stress is a major mechanism of myocardial damage in human CCM. It concurs with calcium overload to myocyte dysfunction and death.
Assuntos
Cardiomiopatia Dilatada/fisiopatologia , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Estresse Oxidativo/fisiologia , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Western Blotting , Cardiomiopatia Dilatada/metabolismo , Catalase/metabolismo , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Feminino , Traumatismos Cardíacos/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estudos Retrospectivos , Superóxido Dismutase/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Adulto JovemRESUMO
BACKGROUND: Cardiac arrhythmias are common in Fabry disease (FD) and may occur in prehypertrophic cardiomyopathy suggesting an early compromise of conduction tissue (CT). Therefore, FD X-linked and CT may be variously involved in male and female patients with FD cardiomyopathy, affecting CT function. METHODS AND RESULTS: Among 74 patients with endomyocardial biopsy diagnosis of FD cardiomyopathy, 13 (6 men; 7 women; mean age, 50.1±13.5 years; maximal wall thickness, 16.7±3.7 mm) had CT included in histological specimens and 6 also at electron microscopy. CT glycolipid infiltration was defined as focal, moderate, extensive, or massive, if involved ≤30%, ≤50%, >50%, or 100% of cells; identified as loosely arranged small myocytes positive to HCN4 immunostaining, supplied by a centrally placed thick-walled arteriole. CT involvement was correlated with age, sex, and α-Gal gene mutation. CT function was evaluated by electrophysiological study and arrhythmias at Holter registration. CT infiltration was focal/moderate in 4 women with no arrhythmias and normal electrophysiological study, extensive in 3 women with atrial or ventricular arrhythmias and short HV interval, and massive in 6 men with atrial fibrillation or ventricular arrhythmias and short HV. Short PR/AH with increased refractoriness was additionally found in 3 patients with extensive/massive CT infiltration. A male patient with the shortest HV presented infra-Hissian block during decremental atrial stimulation. There was no correlation with age, maximal wall thickness, and type of gene mutation. CONCLUSIONS: CT infiltration in FD cardiomyopathy is constant in men and variable in women because of skewed X-chromosome inactivation; its extensive/massive involvement causes accelerated conduction with prolonged refractoriness and electric instability.
Assuntos
Cardiomiopatias/patologia , Técnicas Eletrofisiológicas Cardíacas/métodos , Doença de Fabry/complicações , Átrios do Coração/patologia , Sistema de Condução Cardíaco/ultraestrutura , Miocárdio/patologia , Função Ventricular/fisiologia , Cardiomiopatias/etiologia , Cardiomiopatias/fisiopatologia , Eletrocardiografia , Doença de Fabry/diagnóstico , Doença de Fabry/fisiopatologia , Feminino , Átrios do Coração/fisiopatologia , Frequência Cardíaca/fisiologia , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.
Assuntos
Amônia/farmacologia , Autofagia/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Sirtuínas/metabolismo , Autofagia/fisiologia , Glutaminase/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Proteólise/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Skeletal muscle disturbances are commonly reported in patients with Fabry disease. Whether they derive from cardiac dysfunction or direct muscle involvement is still unclear. Clinical, noninvasive, and invasive cardiac and muscle studies, including an endomyocardial and muscle biopsy, were obtained in 12 patients (mean age, 42.1 ± 12.6 years; range, 24-58 years) with Fabry disease. In the youngest patients (group A, 4 men aged <35 years), results of cardiac and skeletal noninvasive studies were normal, except for reduced velocities in tissue Doppler imaging. Histologic examination indicated that muscle myocytes were unaffected, whereas muscle vessels showed the presence of mild glycosphingolipid accumulation in endothelial and smooth muscle cells. In the heart, cardiomyocytes and endothelial and smooth muscle cells of intramural cardiac vessels were involved by the disease. The oldest patients (group B, 6 men and 2 women aged >35 years) showed ultrasound muscle disarray and electromyography signs of myopathy, increased left ventricular mass, and normal cardiac function. Histologic examination showed that muscle myocytes contained mild glycosphingolipid accumulation compared with severe engulfment of cardiomyocytes. Moreover, similar infiltration of myocardial and muscle intramural vessels, causing lumen narrowing and fibrofatty tissue replacement, was observed. Direct muscle involvement occurs in patients with Fabry disease. It is milder and delayed compared with that in the heart. The difference in organ function and the need of residual α-galactosidase A activity are the likely causes.
Assuntos
Cardiomiopatias/patologia , Doença de Fabry/patologia , Músculo Esquelético/patologia , Miocárdio/patologia , Adulto , Cardiomiopatias/complicações , Cardiomiopatias/fisiopatologia , Doença de Fabry/complicações , Doença de Fabry/fisiopatologia , Feminino , Coração/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Miócitos Cardíacos/patologiaRESUMO
An increasing number of malignancies has been shown to be initiated and propelled by small subpopulations of cancer stem cells (CSC). However, whether tumor aggressiveness is driven by CSC and by what extent this property may be relevant within the tumor mass is still unsettled. To address this issue, we isolated a rare tumor cell population on the basis of its CD44(+)CD24(-) phenotype from the human androgen-independent prostate carcinoma cell line DU145 and established its CSC properties. The behavior of selected CSC was investigated with respect to the bulk DU145 cells. The injection of CSC in nude mice generated highly vascularized tumors infiltrating the adjacent tissues, showing high density of neuroendocrine cells and expressing low levels of E-cadherin and ß-catenin as well as high levels of vimentin. On the contrary, when a comparable number of unsorted DU145 cells were injected the resulting tumors were less aggressive. To investigate the different features of tumors in vivo, the influence of differentiated tumor cells on CSC was examined in vitro by growing CSC in the absence or presence of conditioned medium from DU145 cells. CSC grown in permissive conditions differentiated into cell populations with features similar to those of cells held in aggressive tumors generated from CSC injection. Differently, conditioned medium induced CSC to differentiate into a cell phenotype comparable to cells of scarcely aggressive tumors originated from bulk DU145 cell injection. These findings show for the first time that CSC are able to generate differentiated cells expressing either highly or scarcely aggressive phenotype, thus influencing prostate cancer progression. The fate of CSC was determined by signals released from tumor environment. Moreover, using microarray analysis we selected some molecules which could be involved in this cell-to-cell signaling, hypothesizing their potential value for prognostic or therapeutic applications.
Assuntos
Comunicação Celular/fisiologia , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Animais , Antígeno CD24/metabolismo , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Receptores de Hialuronatos/metabolismo , Masculino , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/metabolismo , Transplante Heterólogo , beta Catenina/metabolismoRESUMO
AIMS: Patients with intestinal malabsorption may develop cardiac dysfunction the origin of which is often unclear. We sought to investigate the pathogenesis of dilated cardiomyopathy in human malabsorption. METHODS AND RESULTS: Eighteen patients with intestinal bypass as treatment for severe obesity and cardiomyopathy underwent endomyocardial biopsy. Biopsies were processed by histology, electron microscopy, polymerase chain reaction (PCR) for cardiotropic viruses, instrumental neutron activation analysis (INAA) of 33 myocardial trace elements, and assessment of glutathione peroxidase (GPX) activity and LC3-II expression. Histology and electron microscopy showed hypertrophy/degeneration of cardiomyocytes with pronounced cell autophagy and high expression of LC3-II. PCR was negative for viral genomes. INAA showed severe myocardial selenium (Se) and zinc (Zn) deficiency and reduced GPX activity vs. both patients with idiopathic dilated cardiomyopathy and normal controls. Se and Zn were added to antifailing heart therapy in 10 patients (group A1) agreeing to a control biopsy, and the response was compared with that of 8 patients (group A2) on supportive therapy alone. After 6 months, myocardial normalization of Se, Zn, LC3-II, and GPX in group A1 was associated with recovery of cardiomyocyte degeneration and autophagy, and significant improvement in cardiac dimension and function, that remained unchanged in group A2. CONCLUSION: A reversible Se- and Zn-deficient cardiomyopathy may occur in patients with intestinal malabsorption. It is characterized by decline of myocardial antioxidant reserve, oxidative damage of cell membranes, and enhanced cell autophagy.
Assuntos
Cardiomiopatia Dilatada/patologia , Deficiências Nutricionais/etiologia , Síndromes de Malabsorção/complicações , Selênio/administração & dosagem , Zinco/administração & dosagem , Adulto , Biópsia , Cardiomiopatia Dilatada/etiologia , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Selênio/análise , Selênio/deficiência , Zinco/análise , Zinco/deficiênciaRESUMO
The highly polarized structure and function of mammalian spermatozoa dictate that these cells compartmentalize specific metabolic and signaling pathways to regions where they are needed. Fas was initially identified as membrane receptor for pro-apoptotic signals, has been recently recognized as a molecule with pleiotropic functions. In this article, we provide evidence of a peculiar Fas localization: it is closely associated to the perinucleus, mainly at the level of the inner acrosomal membrane, as well as in the inner compartment of mitochondria. Immunoelectron microscopy and Western blot analysis indicated that intracellular Fas was associated with mitochondria in mouse epididymal spermatozoa. Accordingly, also in human ejaculated sperm, immunofluorescence analysis showed Fas localized in the middle piece of sperm flagellum where mitochondria are grouped. The potential functional implications of these findings are discussed.
Assuntos
Mitocôndrias/química , Espermatozoides/química , Receptor fas/análise , Animais , Western Blotting , Humanos , Masculino , Camundongos , Microscopia ImunoeletrônicaRESUMO
BACKGROUND: Chest pain is frequently reported in Fabry disease (FD). However, its mechanism and clinical relevance are unclear. METHODS AND RESULTS: Basal troponin I level, exercise stress test, single-photon emission computed tomography imaging with (99m)Tc sestamibi, coronary angiography with thrombolysis in myocardial infarction (TIMI) frame count and left ventricular angiography and endomyocardial biopsy were obtained in 13 patients with FD with angina. Ratio of external to lumen diameter of intramural arteries (E/L ratio), myocyte diameter, and extent of fibrosis were morphometrically evaluated by using tissue sections. Controls for coronary angiography and histology were 25 patients with FD without angina and 20 mitral stenosis patients with normal left ventricular function. Troponin I level was elevated in 6 of the 13 patients. Exercise stress test showed evidence of myocardial ischemia, and single-photon emission computed tomography was positive for stress-induced perfusion defects in all patients with FD with angina. Epicardial coronaries were structurally normal but showed slow flow in all and were associated with aneurisms of posterior left ventricular wall in 3 cases. Histology showed remarkable lumen narrowing of most intramural arteries (mean E/L ratio=3.5+/-1.2; P<0.001 versus both control groups), because of hypertrophy and proliferation of smooth muscle and endothelial cells, both engulfed by glycosphingolipids. Replacement fibrosis exceeded that of both controls (P<0.001). Small vessel disease correlated with coronary slow flow and extent of fibrosis, but did not with patients' age, sex, and degree of left ventricular hypertrophy. CONCLUSIONS: patients with FD with angina have perfusion defects, slow coronary flow, and luminal narrowing of intramural arteries. Small vessel disease may contribute to symptomatic limitation and progressive myocardial dysfunction.
Assuntos
Angina Pectoris/etiologia , Vasos Coronários/patologia , Doença de Fabry/diagnóstico , Adulto , Idoso , Angina Pectoris/diagnóstico , Angina Pectoris/enzimologia , Biópsia , Angiografia Coronária , Diagnóstico Diferencial , Teste de Esforço , Doença de Fabry/complicações , Doença de Fabry/enzimologia , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Leucócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Tomografia Computadorizada de Emissão de Fóton Único , Adulto Jovem , alfa-Galactosidase/sangueRESUMO
It has been reported that sorbitol induces apoptosis in several cancer cell lines. However, the molecular mechanism underlying the sorbitol-induced apoptotic process is not yet clearly understood. In the present study, the intracellular signaling pathways of sorbitol-induced apoptosis in human K562 cells were investigated using both morphological analysis and DNA fragmentation technique. In this study, we demonstrated that sorbitol-induced apoptosis in human K562 cells is a concentration- and time-dependent manner. This sorbitol-induced apoptosis in human K562 cells was also accompanied by the up-regulation of Bax, and down-regulation of p-Bcl-2, but no effect on the levels of Bcl-X(L). Moreover, the sorbitol treatment resulted in a significant reduction of mitochondria membrane potential, increase in the release of mitochondrial cytochrome c (cyt c), and activation of caspase 3. Furthermore, treatment with caspase 3 inhibitor (z-DEVD-fmk) was capable of preventing the sorbitol-induced caspase 3 activity and cell death. These results clearly demonstrate that the induction of apoptosis by sorbitol involves multiple cellular/molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family proteins, mitochondrial membrane potential, mitochondrial cyt c, and caspase 3, they all participate in sorbitol-induced apoptotic process in human K562 cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/biossíntese , Citocromos c/biossíntese , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Sorbitol/farmacologia , DNA/efeitos dos fármacos , Fragmentação do DNA , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-x(L) content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and downregulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17.1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude and did not give rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to control values after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.
Assuntos
Apoptose , Fator de Crescimento Insulin-Like I/metabolismo , Neurônios/citologia , Estaurosporina/farmacologia , Animais , Fator de Indução de Apoptose/metabolismo , Diferenciação Celular , Linhagem Celular , Eletrofisiologia , Endodesoxirribonucleases/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Estaurosporina/antagonistas & inibidores , Proteína X Associada a bcl-2/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Proteína bcl-X/metabolismoRESUMO
Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.