Navegando por Palavras-chave "Cell death"

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    Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells
    (Elsevier B.V., 2014-07-01) Silva, Mariana C. C. [UNIFESP]; Paula, Claudia A. A. de [UNIFESP]; Ferreira, Joana G. [UNIFESP]; Paredes-Gamero, Edgar Julian [UNIFESP]; Vaz, Angela M. S. F.; Sampaio, Misako U. [UNIFESP]; Correia, Maria Tereza S.; Oliva, Maria Luiza V. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst Pesquisas Jardim Bot Rio de Janeiro; Universidade Federal de Pernambuco (UFPE)
    Background: Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. in this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp.forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity.Methods: MCF7 cells were treated with BfL Viability and adhesion alterations were evaluated using flow cytometry and western blotting.Results: BfL. inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases alpha(1), alpha(6) and beta(1) integrin subunit expression, and increases alpha(5) subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic add (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21.Conclusion: BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells. General significance: Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer. (C) 2014 Elsevier B.V. All rights reserved.
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    Bcl-X-L inhibits Bax-induced alterations in mitochondrial respiration and calcium release
    (Elsevier B.V., 2008-09-12) Teles, Alessandra Vaz Fernandes Fiuza [UNIFESP]; Ureshino, Rodrigo Portes [UNIFESP]; Dorta, Daniel Junqueira [UNIFESP]; Lopes, Guiomar Silva [UNIFESP]; Hsu, Yi-Te; Smaili, Soraya Soubhi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Med Univ S Carolina
    Apoptosis is a natural cell elimination process involved in a number of physiological and pathological events. This process can be regulated by members of the Bcl-2 family. Bax, a pro-apoptotic member of this family, accelerates cell death, while the pro-survival member, Bcl-X-L, can antagonize the pro-apoptotic function of Bax to promote cell survival. in the present study, we have evaluated the effect of Bcl-X-L on Bax-induced alterations in mitochondrial. respiration and calcium release. We found that in primary cultured astrocytes, recombinant Bcl-X-L is able to antagonize Bax-induced decrease in mitochondrial respiration and increase in mitochondrial. calcium release. in addition, we found that Bcl-X-L can lower the calcium store in the endoplasmic reticulum, thus limiting potential calcium flux induced by apoptosis. This regulation of calcium flux by Bcl-X-L may represent an important mechanism by which this protein promotes cell survival. (c) 2008 Elsevier Ireland Ltd. All rights reserved.
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    Beta carbolinas: estudo dos mecanismos de morte, proliferação e diferenciação em leucemias e células-tronco leucêmicas
    (Universidade Federal de São Paulo (UNIFESP), 2018-10-25) Torquato, Heron Fernandes Vieira [UNIFESP]; Gamero, Edgar Julian Paredes [UNIFESP]; http://lattes.cnpq.br/9562213378846769; http://lattes.cnpq.br/7439686090439905; Universidade Federal de São Paulo (UNIFESP)
    Acute myeloid leukemia ( AML) is a type of cancer that occurs from the clonal expansion of myeloid pr ecursors. Over the past few years, a hypothesis used to explain the leukemia origin has been the presence of a rare population inside of the tumor, which represents a refractory reservoir to therapy, named leukemic stem cell (LSC). For this reason, the elimination of this population is considered an important treatment method nowadays. Among all natural products that have provided new molecules with different clinical applications, we highlight the betacarbolinic alkaloids, in particular, the canthinone and its derived, which have demonstrated diverse pharmacological activities s uch as antibacterial, antifungal, antimalarial, antiulcerogenic and cytotoxic. Thus, the aim of this project was to investigate the antitumor effects of betacarbolinic alkaloids in leukemias and in the LSC population. Therefore, the effects of betacarboline alkaloids on cell death, proliferation and differentiation were investigated. It was observed that the effect on cell death occurred by apoptosis ( dissipation of mitochondrial potential, permeabilization of lysosomes, production of reactive oxygen species [ROS], and activation of caspases) and by Necroptosis ( use of pharmacological inhibitors of necroptosis, Nec1 and necrosulfonamide, as well as their association with caspase inhibitors). In addition, a cytostatic effect with cell cycle ar rest in the G2/M phase associated by activation of DNA damage sensor proteins and the decrease of clonogenic capability was observed. Induction of cell differentiation was also observed by expression increased of myeloid differentiation markers ( CD15, C D11b, C D14, P U.1 and MPO). The differentiation also affected LSC population with expression increase of PU.1 factor, and proliferation increase, but the reduction of clonogenic potential. Part of the effects on differentiation and proliferation was blocked by the p38 inhibitor, SB203580. The results show important actions of this metabolites class, highlighting the effects of MTXc as privileged structures for the construction of new antileukemic drugs
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    BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity
    (Academic Press Inc Elsevier Science, 2016) Ferreira, Adilson Kleber; Mesquita Pasqualoto, Kerly Fernanda; Kruyt, Frank A. E.; Palace-Berl, Fanny; Azevedo, Ricardo Alexandre; Turra, Kely Medeiros; Rodrigues, Cecilia Pessoa; Franco Ferreira, Ana Carolina; Clavijo Salomon, Maria Alejandra; de Sa Junior, Paulo Luiz; Farias, Camyla Fernandes [UNIFESP]; Figueiredo, Carlos Rogerio [UNIFESP]; Tavares, Leoberto Costa; Marzagdo Barbuto, Jose Alexandre; Jorge, Salomao Doria
    Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MIT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma. (C) 2016 Elsevier Inc. All rights reserved.
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    Bothropoides insularis venom cytotoxicity in renal tubular epithelia cells
    (Elsevier B.V., 2014-09-15) Mello, Clarissa P.; Morais, Isabel C. O.; Menezes, Ramon R. P. P. B.; Pereira, Gustavo J. S. [UNIFESP]; Torres, Alba F. C.; Lima, Danya B.; Pereira, Ticiana P.; Toyama, Marcos H.; Monteiro, Helena S. A.; Smaili, Soraya Soubhi [UNIFESP]; Martins, Alice M. C.; Univ Fed Ceara; Universidade Federal de São Paulo (UNIFESP); Univ Estadual Paulista
    Bothropoides insularis (jararaca-ilhoa) is a native endemic snake limited to the specific region of Queimada Island, on São Paulo coast. Several local and systemic effects have been described due to envenomation caused by it, such as edema, tissue necrosis, hemorrhage and acute renal failure. Our previous studies have shown that Bothropoides insularis venom (BinsV) demonstrated important functional and morphologic alterations in rat isolated kidney, especially decrease in tubular electrolyte transport, osmotic clearance and tubular necrosis. in order to elucidate the direct nephrotoxicity mechanism, the aim of the present study was to investigate BinsV cytotoxicity effect on renal epithelial cells. the treatment with BinsV over MDCK culture decreased cell viability in all concentrations tested with IC50 of 9 mu g/mL. BinsV was able to induce membrane rupture and cell death with phosphatidilserine externalization. Furthermore, BinsV induced ROS overproduction and mitochondrial membrane potential collapse, as well as Bax translocation and caspases 3 and 7 expression. Therefore, these events might be responsible by BinsV-induced cell death caused by mitochondrial dysfunction and ROS overproduction in the direct cytotoxicity process. (C) 2014 Elsevier B.V. All rights reserved.
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    Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells
    (Elsevier Science Bv, 2017) Vieira Torquato, Heron F. [UNIFESP]; Ribeiro-Filho, Antonio C.; Buri, Marcus V. [UNIFESP]; Araujo Junior, Roberto T. [UNIFESP]; Pimenta, Renata [UNIFESP]; de Oliveira, Jose Salvador R. [UNIFESP]; Filho, Valdir C.; Macho, Antonio; Paredes-Gamero, Edgar J. [UNIFESP]; de Oliveira Martins, Domingos T.
    Background: Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Methods: Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin(+)) and leukemia stem cell population (CD34(+) CD38(-)Lin(-/low) Results: Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at Go/Gi (7 mu M) and G2 (45 mu m) evidenced by DNA content, BrdU incorporation and cyclin Bl/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11 b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. Conclusions: These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. General significance: Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. (C) 2017 Elsevier B.V. All rights reserved.
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    Characterization of dual effects induced by antimicrobial peptides: Regulated cell death or membrane disruption
    (Elsevier B.V., 2012-07-01) Paredes-Gamero, Edgar Julian [UNIFESP]; Martins, Marta Natividade Crizol [UNIFESP]; Cappabianco, Fabio Augusto Menocci [UNIFESP]; Ide, Jaime Shinsuke [UNIFESP]; Miranda, Antonio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Background: Some reports describe lysis mechanisms by antimicrobial peptides (AMPs), while others describe the activation of regulated cell death. in this study, we compare the cell death-inducing activities of four beta-hairpin AMPs (gomesin, protegrin, tachyplesin and polyphemusin II) along with their linear analogs in the human erythroleukemia K562 cell line to investigate the relationship between their structure and activity.Methods: K562 cells were exposed to AMPs. Morphological and biochemistry alterations were evaluated using light microscopy, confocal microscopy and flow cytometry.Results: Gomesin and protegrin displayed cytotoxic properties that their linear counterparts did not. Tachyplesin and polyphemusin II and also their linear analogs induced cell death. We were able to distinguish two ways in which these AMPs induced cell death. Lower concentrations of AMPs induced controlled cell death mechanisms. Gomesin, tachyplesin and linear-tachyplesin promoted apoptosis that was characterized by annexin labeling, sensitivity to Z-VAD, and caspase-3 activation, but was also inhibited by necrostatin-1. Gomesin and protegrin induced cell death was dependent on intracellular Ca2+ mechanisms and the participation of free radicals was observed in protegrin induced cell death. Polyphemusin II and its linear analog mainly induced necrosis. Conversely, treatment with higher concentrations of AMPs primarily resulted in cell membrane disruption, but with clearly different patterns of action for each AMP tested.Conclusion: Different actions by beta-hairpin AMPs were observed at low concentrations and at higher concentrations despite the structure similarity.General significance: Controlled intracellular mechanism and direct membrane disruption were clearly distinguished helping to understand the real action of AMPs in mammalian cells. (C) 2012 Elsevier B.V. All rights reserved.
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    Cimetidine-induced vascular cell apoptosis impairs testicular microvasculature in adult rats
    (F Hernandez, 2012-10-01) Beltrame, Flavia Luciana [UNIFESP]; Yamauti, Caroline T.; Caneguim, Breno Henrique [UNIFESP]; Cerri, Paulo Sergio [UNIFESP]; Miraglia, Sandra Maria [UNIFESP]; Sasso-Cerri, Estela [UNIFESP]; Sao Paulo State Univ UNESP; Universidade Federal de São Paulo (UNIFESP)
    Cimetidine, an H-2 receptor antagonist used for treatment of gastric ulcers, exerts antiandrogenic and antiangiogenic effects. In the testes cimetidine impairs spermatogenesis, Sertoli cells and peritubular tissue, inducing apoptosis in the myoid cells. Regarding the importance of histamine and androgens for vascular maintenance, the effect of cimetidine on the structural integrity of the testicular vasculature was evaluated. Adult male rats received cimetidine (CMTG) and saline (CG) for 50 days. The testes were fixed in buffered 4% formaldehyde and embedded in historesin and paraffin. In the PAS-stained sections, the microvascular density (MVD) and the vascular luminal area (VLA) were obtained. TUNEL method was performed for detection of cell death. Testicular fragments embedded in Araldite were analyzed under transmission electron microscopy. A significant decrease in the MVD and VLA and a high number of collapsed blood vessel profiles were observed in CMTG. Endothelial cells and vascular muscle cells were TUNEL-positive and showed ultrastructural features of apoptosis. These results indicate that cimetidine induces apoptosis in vascular cells, leading to testicular vascular atrophy. A possible antagonist effect of cimetidine on the H-2 receptors and/or androgen receptors in the vascular cells may be responsible for the impairment of the testicular microvasculature.
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    Consequences of pilocarpine-induced status epilepticus in immunodeficient mice
    (Elsevier B.V., 2012-04-23) Vignoli, Thiago [UNIFESP]; Nehlig, Astrid; Massironi, Silvia Gomes; Sinigaglia Coimbra, Rita de Cassia [UNIFESP]; Naffah Mazzacoratti, Maria da Graca [UNIFESP]; Silva, Iara Ribeiro [UNIFESP]; Castro Neto, Eduardo Ferreira de [UNIFESP]; Persike, Daniele Suzete [UNIFESP]; Silva Fernandes, Maria Jose da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Strasbourg; Universidade de São Paulo (USP)
    Systemic injection of pilocarpine in rodents induces status epilepticus (SE) and reproduces the main characteristics of temporal lobe epilepsy (TLE). Different mechanisms are activated by SE contributing to cell death and immune system activation. We used BALB/c nude mice, a mutant that is severely immunocompromised, to characterize seizure pattern, neurochemical changes, cell death and c-Fos activation secondarily to pilocarpine-induced SE. the behavioral seizures were less severe in BALB/c nude than in BALB/c wild type mice. However, nude mice presented more tonic clonic episodes and higher mortality rate during SE. the c-Fos expression was most prominent in the caudate-putamen, CA3 (p < 0.05), dentate gyrus, entorhinal cortex (p < 0.001), basolateral nucleus of amygdala (p < 0.01) and piriform cortex (p < 0.05) of BALB/c nude mice than of BALB/c. Besides, nude mice subjected to SE presented high number of Fluorojade-B (FJB) stained cells in the piriform cortex, amygdala (p < 0.05) and hilus (p < 0.05) in comparison with BALB/c mice. A significant increase in the level of glutamate and GABA was found in the hippocampus and cortex of BALB/c mice presenting SE in comparison to controls. However, the level of glutamate was higher in the brains of BALB nude mice than in the brains of BALB/c wild type mice, while the levels of GABA were unchanged. These results indicate that the brains of immunodeficient nude mice are more vulnerable to the deleterious effects of pilocarpine-induced SE as they present intense activation, increased glutamate levels and more cell death. Published by Elsevier B.V.
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    Control of infection by pyroptosis and autophagy: role of TLR and NLR
    (Springer, 2010-05-01) Bortoluci, Karina R. [UNIFESP]; Medzhitov, Ruslan; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Howard Hughes Med Inst; Yale Univ
    Cells can die by distinct mechanisms with particular impacts on the immune response. in addition to apoptosis and necrosis, recent studies lead to characterization of a new pro-inflammatory form of cell death, pyroptosis. TLR and NLR, central innate immune sensors, can control infections by modulating host cell survival. in addition, TLRs can promote the induction of autophagy, thus promoting delivery of infecting pathogens to the lysosomes. On the other hand, activation of some NLR members, especially NLRC4 and NAIP5, leads to the infected cell death by pyroptosis, which is accompanied by secretion of the pro-inflammatory cytokines IL-1 beta, IL-18, and IL-33. Data presented here illustrate how the compartmentalization of the innate immune sensors can influence the outcome of infections by controlling the fate of host cells.
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    Cytotoxicity of phenothiazine derivatives associated with mitochondrial dysfunction: A structure-activity investigation
    (Elsevier B.V., 2015-04-01) Faria, Priscila A. de; Bettanin, Fernanda; Cunha, Rodrigo L. O. R.; Paredes-Gamero, Edgar J. [UNIFESP]; Homem-de-Mello, Paula; Nantes, Iseli L.; Rodrigues, Tiago; Universidade Federal do ABC (UFABC); Universidade Federal de São Paulo (UNIFESP)
    Phenothiazine derivatives are neuroleptic drugs used in the treatment of schizophrenia and anxiety. Several side effects are described for these drugs, including hepatotoxicity, which may be related to their cytotoxic activity. Working with isolated rat liver mitochondria, we previously showed that phenothiazine derivatives induced the mitochondrial permeability transition associated with cytochrome c release. Since the mitochondrial permeabilization process plays a central role in cell death, the aim of this work was to evaluate the effects of five phenothiazine derivatives (chlorpromazine, fluphenazine, thioridazine, trifluoperazine, and triflupromazine) on the viability of hepatoma tissue culture (HTC) cells to establish the structural requirements for cytotoxicity. All phenothiazine derivatives decreased the viability of the HTC cells in a concentration-dependent manner and exhibited different cytotoxic potencies. the EC50 values ranged from 45 to 125 mu M, with the piperidinic derivative thioridazine displaying the most cytotoxicity, followed by the piperazinic and aliphatic derivatives. the addition of the phenothiazine derivatives to cell suspensions resulted in significant morphological changes and plasma membrane permeabilization. Octanol/water partition studies revealed that these drugs partitioned preferentially to the apolar phase, even at low pH values (>= 4.5). Also, structural and electronic properties were calculated employing density functional theory. Interestingly, the phenothiazine derivatives promoted an immediate dissipation of the mitochondrial transmembrane potential in HTC cells, and the EC50 values were closely correlated with those obtained in cell viability assays, as well as the EC50 for swelling in isolated mitochondria. These results significantly contribute to improving our understanding of the specific structural requirements of the phenothiazine derivatives to induce cell death and suggest the involvement of the mitochondrial permeability transition in phenothiazine-induced cytotoxicity in HTC cells. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
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    Different patterns of neuronal activation and neurodegeneration in the thalamus and cortex of epilepsy-resistant Proechimys rats versus Wistar rats after pilocarpine-induced protracted seizures
    (Wiley-Blackwell, 2009-04-01) Andrioli, Anna; Fabene, Paolo F.; Spreafico, Roberto; Cavalheiro, Esper Abrão [UNIFESP]; Bentivoglio, Marina; Univ Verona; Neurol Inst Carlo Besta; Universidade Federal de São Paulo (UNIFESP)
    To analyze cellular mechanisms of limbic-seizure suppression, the response to pilocarpine-induced seizures was investigated in cortex and thalamus, comparing epilepsy-resistant rats Proechimys guyannensis with Wistar rats.Fos immunoreactivity revealing neuronal activation, and degenerating neurons labeled by Fluoro-Jade B (FJB) histochemistry were analyzed on the first day after onset of seizures lasting 3 h. Subpopulations of gamma-aminobutyric acid (GABA)ergic cells were characterized with double Fos-parvalbumin immunohistochemistry.In both cortex and thalamus, degenerating neurons were much fewer in Proechimys than Wistar rats. Fos persisted at high levels at 24 h only in the Proechimys thalamus and cortex, especially in layer VI where corticothalamic neurons reside. in the parietal cortex, about 50% of parvalbumin-containing interneurons at 8 h, and 10-20% at 24 h, were Fos-positive in Wistar rats, but in Proechimys, Fos was expressed in almost all parvalbumin-containing interneurons at 8 h and dropped at 24 h. Fos positivity in cingulate cortex interneurons was similar in both species. in the Wistar rat thalamus, Fos was induced in medial and midline nuclei up to 8 h, when < 30% of reticular nucleus cells were Fos-positive, and then decreased, with no relationship with cell loss, evaluated in Nissl-stained sections. in Proechimys, almost all reticular nucleus neurons were Fos-positive at 24 h.At variance with laboratory rats, pilocarpine-induced protracted seizures elicit in Proechimys limited neuronal death, and marked and long-lasting Fos induction in excitatory and inhibitory cortical and thalamic cell subsets. the findings implicate intrathalamic and intracortical regulation, and circuits linking thalamus and cortex in limbic seizure suppression leading to epilepsy resistance.
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    Efeito do condroitim sulfato na fibrogênese hepática induzida por ligadura do ducto biliar em ratos
    (Universidade Federal de São Paulo (UNIFESP), 2017-10-26) Guedes, Pedro Luiz Rodrigues [UNIFESP]; Nagaoka, Márcia Regina [UNIFESP]; http://lattes.cnpq.br/3554142919645884; http://lattes.cnpq.br/4997715000909196; Universidade Federal de São Paulo (UNIFESP)
    Introdução: Uma das causas de fibrose hepática é a colestase, que causa morte celular e desencadeia a liberação de citocinas e quimiocinas, levando à infiltração de leucócitos e à deposição de matriz extracelular. O tratamento atual da fibrose hepática é baseado na retirada do agente causador, por isso é importante a investigação de estratégias novas e eficazes para o tratamento da fibrose hepática. Muitos estudos destacam a ação do condroitim sulfato em diferentes tipos de células, com consequente redução da expressão de citocinas. O objetivo deste trabalho foi analisar os efeitos do condroitim sulfato (CS) sobre o modelo experimental de colestase extra-hepática induzida pela ligadura do ducto biliar comum (BDL). Metodologia: Ratos Wistar (6-8 semanas) foram submetidos a BDL e receberão injeção intraperitoneal de CS (120 mg/kg peso) ou veículo por 1, 2, 7, 14, 21 ou 28 dias; os animais sham foram utilizados como controle. Ao final do período de tratamento, os animais foram eutanasiados e amostras de fígado e sangue retiradas. Foram analisadas as atividades de alanina e aspartato aminotransferases séricas (ALT e AST, respectivamente) e, caspase-3 e catepsina B em homogenatos de fígado utilizando os substratos fluorométricos Ac-DEVD-AMC e Abz-GIVRAK(Dnp)-OH, respectivamente. Em espécimes de fígado foi realizada análise morfométrica usando o programa Axiovision 4,8, além de avaliar a apoptose por reação de TUNEL e regeneração hepática por imuno-histoquímica para o antígeno nuclear de proliferação celular (PCNA).. Os resultados foram expressos pela média ± EPM. A análise estatística de valores padronizados foi realizada por ANOVA unidirecional com post-hoc de Tukey. Resultados: Animais BDL apresentaram atividades séricas de ALT e AST 2-3 vezes maiores do que os sham. BDL levou ao progressivo desenvolvimento de septos fibróticos, confirmado pela área de colágeno (%) após 7 (11 ± 1, n = 7), 14 (14 ± 1, n = 10), 21 (23 ± 1, n = 4) e 28 (34 ± 3, n = 7) dias de indução, enquanto os grupos sham tiveram a arquitetura normal do fígado preservada. O tratamento com CS reduziu o conteúdo de colágeno após 21 dias (14 ± 1, n = 6) e significativamente (p <0,001) após 28 dias (16 ± 2, n = 6) em relação ao não-tratado. Não foram observadas alterações significativas nas atividades da catepsina B de todos os grupos estudados. Verificou-se aumento progressivo da atividade da caspase-3 (relativo ao sham) no grupo BDL 2d (1,4 ± 0,1, n = 5), BDL 7d (2,2 ± 0,6, n = 7) e 14d (3,3 ± 0,5, n = 10; ANOVA, p < 0,001) quando comparados aos respectivos grupos sham. Interessante, CS reduziu significativamente (p= 0,024) a atividade de caspase-3 após 14 dias de tratamento (1,7 ±0,7, n = 9) quando comparado com o grupo BDL 14d. Os animais BDL apresentaram mais células apoptóticas por campo (% do total) aos 14 dias (0,58±0,04) do que aos 7 (0,25±0,02), 21 (0,33±0,02) e 28 dias (0,29±0,01). O tratamento com CS reduziu significativamente (p = 0,030) a quantidade de células em apoptose nos animais 14d (0,43±0,04). BDL levou ao aumento significativo na quantidade relativa (%) de células epiteliais biliares e de hepatócitos com núcleo marcado com anti-PCNA. O tratamento com CS levou ao aumento significativo de hepatócitos em proliferação (No relativo) nos animais 14d (2,9±0,1 – ANOVA; p = 0,003) e 21d (3,5±0,2 - ANOVA; p < 0,001) quando comparados aos respectivos BDL não tratados (14d: 2,1±0,1 e 21d: 2,2±0,21). Conclusão: Este trabalho mostra que a CS pode reduzir os primeiros sinais de apoptose, retardar o desenvolvimento de fibrose hepática e, portanto, cirrose, além de promover a regeneração hepática em modelo experimental de ligadura do ducto biliar.
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    Efeitos da inibição da via autofágica na indução da apoptose e na síntese de espécies reativas de oxigênio em oócitos bovinos expostos à hiperglicemia
    (Universidade Federal de São Paulo, 2022-01-27) Reis, Veronica Louzada [UNIFESP]; Lopes, Fabíola Freitas de Paula [UNIFESP]; http://lattes.cnpq.br/0954914266701996; http://lattes.cnpq.br/0604096975417271
    O diabetes mellitus (DM) é uma das enfermidades que mais acomete a população humana. A hiperglicemia induzida durante o DM provoca severos danos ao sistema reprodutor. Os oócitos apresentam maior propensão a desbalanços metabólicos ou danos ambientais durante a maturação e são excelentes modelos para investigação dos efeitos da hiperglicemia. Já foi demonstrado que concentrações de glicose acima de ~10 mM no meio de maturação in vitro ativa mecanismos de morte celular, como a apoptose, e induz a formação de espécies reativas de oxigênio (EROs). Sabe-se que a autofagia atua como um mecanismo de sobrevivência em condições de estresse, entretanto, pouco se sabe sobre os efeitos deletérios da inibição da autofagia em células germinativas expostas à hiperglicemia. Dessa forma, este projeto fez uso do sistema de maturação in vitro (MIV) de oócitos bovinos como modelo a fim de determinar o papel da autofagia durante a MIV de oócitos expostos à hiperglicemia na síntese de EROs, na progressão meiótica e na indução de apoptose oocitária. Portanto, complexos cumulus-oócitos (CCOs) foram incubados com o inibidor da autofagia (0 ou 10 mM 3-metiladenina – 3-MA) em meio contendo concentrações de 5,5 (grupo controle) ou 20 mM (grupo hiperglicêmico) de glicose durante 21 horas de MIV. Os CCOs foram desnudados após a MIV e dividido em dois grupos. Para o primeiro experimento, parte dos oócitos foi utilizada para determinação de células apoptóticas pelo ensaio de TUNEL e análise da progressão meiótica para metáfase II (MII). A hiperglicemia e a inibição da autofagia não afetaram a porcentagem de células apoptóticas. No entanto, a presença de 3-MA na MIV reduziu a porcentagem de oócitos em MII nos grupos com 5,5 mM (P= 0,0312) e 20 mM de glicose (P= 0,01410). O segundo experimento determinou os níveis de EROs nos oócitos a partir do ensaio Cell ROX Green. A hiperglicemia aumentou a síntese de EROs em comparação aos demais grupos (P= 0,0029). Além disso, houve uma tendência de aumento da produção de EROs no grupo hiperglicêmico com 3-MA (P= 0,0769). Os resultados indicaram que os oócitos foram comprometidos pela hiperglicemia por meio do aumento do estresse oxidativo e pela inibição da autofagia, reduzindo a maturação nuclear. É possível que esses danos celulares comprometam a qualidade oocitária, reduzindo a fertilidade.
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    Endoplasmic Reticulum Calcium Release Engages Bax Translocation in Cortical Astrocytes
    (Springer, 2011-05-01) Morales, A. P.; Carvalho, A. C. P.; Monteforte, P. T.; Hirata, H.; Han, S. W. [UNIFESP]; Hsu, Y. -T.; Smaili, Soraya Soubhi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Med Univ S Carolina
    Apoptosis is a highly complex form of cell death that can be triggered by alterations in Ca(2+) homeostasis. Members of the Bcl-2 family may regulate apoptosis and modulate Ca(2+) distribution within intracellular compartments. Bax, a proapoptotic member of the family, is constitutively expressed and soluble in the cytosol and, under apoptotic induction, translocates to mitochondrial membranes. However, it is not clear if the intracellular Ca(2+) stores and selective Ca(2+) releases can modulate or control Bax translocation. the aim of this study was to investigate the relation of intracellular Ca(2+) stores with Bax translocation in rat cortical astrocytes. Results show that the classical apoptotic inducer, staurosporine, caused high elevations of cytosolic Ca(2+) that precede Bax translocation. On the other hand, agents that mobilize Ca(2+) from endoplasmic reticulum such as noradrenaline or thapsigargin, induced Bax translocation, while mitochondrial Ca(2+) release evoked by carbonyl cyanide-p-(trifluoromethoxyphenyl) hydrazone was not able to cause Bax punctation. in addition, microinjection of inositol 1,4,5- trisphosphate induced Bax translocation. Taken together, our results show that in Bax overexpressing cortical astrocytes, endoplasmic reticulum-Ca(2+) release may induce Bax transactivation and specifically control apoptosis.
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    Estudo da autofagia e dos mecanismos de morte após privação nutricional para quimiosensibilização de modelos de melanoma humano Wild Type e Braf V600e
    (Universidade Federal de São Paulo (UNIFESP), 2018-06-28) Antunes, Fernanda [UNIFESP]; Smaili, Soraya Soubhi [UNIFESP]; http://lattes.cnpq.br/6368730022418127; http://lattes.cnpq.br/4094866581034271; Universidade Federal de São Paulo (UNIFESP)
    O melanoma metastático, embora seja o menos prevalente dentre os tipos de câncer de pele, é o mais letal por ser extremamente resistente aos tratamentos atualmente disponíveis, tornando a busca por novas abordagens terapêuticas urgentemente necessárias. Embora a exposição à radiação UV represente a principal causa da melanomagênese, estudos recentes evidenciam também a presença de várias mutações genéticas, como BRAFV600E, presente em aproximadamente 70% dos melanomas, e que promove a ativação constitutiva da via RASRAFMAPK, levando à proliferação celular descontrolada. Esta mutação também está relacionada à inativação de vias de morte celular, desregulação de autofagia, indução de estresse de retículo e favorecimento do metabolismo glicolítico. que coletivamente resultam em resistência a qualquer tratamento. Recentemente foi demonstrado que alterações na dieta podem influenciar tanto o desenvolvimento como o tratamento de diversos tipos de câncer. Um dos protocolos em estudo consiste em ciclos de restrição severa de nutrientes associados a agentes antitumorais. Entretanto, os mecanismos moleculares responsáveis pelos efeitos quimiossensibilizantes ainda não foram completamente elucidados. Baseado nessas premissas, o objetivo geral deste trabalho foi estudar os efeitos da privação nutricional em combinação com terapias antitumorais para o tratamento do melanoma metastático. Para tanto, linhagens de melanoma humano BRAFWT e BRAFV600E e melanoma murino foram submetidas à avaliação qualiquantitativa de morte celular após privação nutricional e/ou tratamento com cisplatina ou sorafenibe. Observouse que, independetemente do status mutacional de BRAF, a privação nutricional sensibilizou as linhagens de melanoma aos tratamentos antitumorais. A morte celular foi predominantemente apoptótica, com determinante participação da mitocôndria no processo de morte celular. Além disso, o bloqueio da glicólise potencializou os efeitos da privação nutricional associada tanto à cisplatina como ao sorafenibe. Adicionalmente, a privação nutricional e os tratamentos antitumorais, isoladamente, induziram autofagia. No entanto, quando associada privação nutricional e sorafenibe, houve bloqueio dos estágios finais do processo autofágico, que assim como o cotratamento com cloroquina, sensibilizou as linhagens de melanoma à morte celular. Já a análise in vivo demonstrou que o uso de intermittent fasting foi seguro e efetivo em reduzir o crescimento tumoral quando associado ao tratamento com sorafenibe. Além disso, foi observada indução de autofagia nos tecidos tumoral e hepático após intermitente fasting e sorafenibe, mas não na sua associação. Coletivamente, nossos resultados indicam que a privação nutricional em associação a agentes antitumorais pode representar uma nova abordagem terapêutica no tratamento do melanoma metastático tanto BRAFWT como BRAFV600E.
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    Estudo da sinalização da morte celular e de alterações comportamentais em modelos animais da doença de Huntington
    (Universidade Federal de São Paulo (UNIFESP), 2004) Rosenstock, Tatiana Rosado [UNIFESP]; Smaili, Soraya Soubhi [UNIFESP]
    Introdução: Alterações na homeostase do Ca2+ podem levar a desordens neurodegenerativas como a Doença de Huntington (DH). A DH e caracterizada por declínio cognitivo, distúrbio psíquico e movimentos motores involuntários. Vários mecanismos, além do genético, tem sido sugeridos como responsáveis: excitotoxicidade, estresse oxidativo e disfunção mitocondrial, que envolve transição de permeabilidade mitocondrial (TPM), colapso do potencial de membrana mitocondrial (,NIY,) e liberação do cálcio mitocondrial (Ca2+m). Juntos, esses fatores podem levar a apoptose. Objetivo: O objetivo deste projeto foi investigar os mecanismos celulares envolvendo o transporte de Ca 2+ relacionados com a disfunção mitocondrial e a morte celular, bem como a relação destes mecanismos com os desvios comportamentais, em dois modelos animais da DH: a) animais tratados com o ácido 3-nitropropionico (3NP), inibidor da enzima succinato desidrogenase (SDH), componente da cadeia respiratória mitocondrial; b) animais transgênicos da linhagem R6/1. Conclusões: As alterações comportamentais induzidas pelo 3NP parecem estar relacionadas com a inibição mitocondrial e com o aumento do estresse oxidativo, diminuição ∆m, liberação do Ca 2'm e morte celular. As fatias cerebrais dos animais transgênicos apresentaram alterações na homeostase de Ca 2+ que podem estar relacionadas a degeneração observada na DH. Por outro lado, os transgênicos não apresentaram diferença quanto a SDH. Esse resultado pode ser devido a idade dos animais, e não descarta a possibilidade de tais diferenças aparecerem em uma fase tardia da DH. Além disso, nossos resultados mostraram que os animais transgênicos possuem uma tendência a menor atividade geral, maior movimentos mandibulares e maior alteração de equilíbrio e coordenação motora, bem como um déficit significante de memória
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    Estudos dos efeitos de concentrações crescentes de óxido nitrico no processo de morte por perda de adesão ao substrato (Anoikis): o papel desempenhado no processo pela proteína Src quinase
    (Universidade Federal de São Paulo (UNIFESP), 2017-05-30) Costa, Paulo Eduardo da [UNIFESP]; Monteiro, Hugo Pequeno [UNIFESP]; http://lattes.cnpq.br/6154759166234850; http://lattes.cnpq.br/2047870426131771; Universidade Federal de São Paulo (UNIFESP)
    The study of cell death induced by loss of adhesion (anoikis) is essential to understand how metastatic tumor cells may develop resistance to this type of cell death. Among a number of factors that can influence anoikis, nitric oxide has your role little known in relation to this issue. In adhered cells, it is known that nitric oxide induces cellular proliferation at low concentrations, as well as induces cell death after the treatment with high concentrations. However, in our study, we observed that detached cells behave in a different mode when treated with increase concentrations of NO: Low concentrations of NO induces drop on cell viability in detached HeLa cells, as well as increase levels of cleaved caspase-3. Since high concentrations of NO induces anoikis protection, with increase on cell viability, decreased of caspase-3 cleavage and decrase of expression of Bim protein. Src kinase has been shown to have key role in this mechanism, being phosphorylated and nitrosylated following treatment with high concentrations of NO. Inhibition of Src, can reverse this protection induced by high concentrations of NO with respect to cell viability and expression of Bim protein.These viability results were reproduced in Nex8H metastatic melanomas, but not in non metastatic melanomas Nex10C. In HeLa cells kept in suspension, NO in high concentrations induced cell disaggregation in a Src dependent manner. NO-dependent disaggregation can be reproduced in murine melanoma cells Nex8H and Nex10C. Increasing concentrations of NO decreased the effectiveness of reattachment of HeLa cells kept in suspension. Src kinase showed no role in the process. We know that tumor cells often produce high concentrations of NO. This study shows up as a first step in understanding how these cells can migrate from a primary to a secondary source and got to readhere regarding the role of increasing concentrations of NO in the process.
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    Evaluation of pyroptosis in macrophages using cytosolic delivery of purified flagellin
    (Elsevier B.V., 2013-06-01) Lage, Silvia L. [UNIFESP]; Amarante-Mendes, Gustavo P.; Bortoluci, Karina R. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); INCT; INCTV
    Pyroptosis is a molecularly controlled form of cell death that exhibits some features of apoptosis as well of necrosis. Pyroptosis is induced by inflammasome-activated caspase-1 or caspase-11 (caspase-4 in humans), as a result of distinct pathogenic or damage stimuli. Although pyroptosis displays some morphological and biochemical features of apoptosis, it has an inflammatory outcome due to the loss of plasma membrane integrity and the consequent release of intracellular contents, reminiscent to necrosis. Here, we use cytosolic delivery of purified flagellin as an experimental tool to trigger pyroptosis and describe potential methods to study this form of cell death. Finally, we discuss the advantages and limitations of these methods. (C) 2013 Elsevier Inc. All rights reserved.
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    Glicogênio sintase quinase 3 e autofagia como alvos moleculares do carbonato de lítio e ácido ursólico em leucemias: possível aumento da atividade da Ara-c
    (Universidade Federal de São Paulo (UNIFESP), 2016-08-31) Santos, Caroline Palmeira dos [UNIFESP]; Trindade, Claudia Bincoletto [UNIFESP]; http://lattes.cnpq.br/6169006634951828; http://lattes.cnpq.br/7711760196963043; Universidade Federal de São Paulo (UNIFESP)
    Objectives: To evaluate the molecular mechanisms involved in the increased cytotoxic effects of Ara-C for lithium carbonate (LIT) in leukemic cells HL-60, and examine possible antileukemic properties of ursolic acid (URS), firstly alone, aiming studies in combination therapy with Ara-C. Methods: The leukemic lines HL-60 and NB4 were used as study models in the following tests: MTT reduction; incorporation of trypan blue dye; evaluation of cell death by labeling fragmented DNA; analysis of the cell cycle phases with propidium iodide; evaluation of modalities of cell death by simultaneous labeling with propidium iodide (PI) and annexin-V-FITC; semi- quantitation of the p62, LC3-II PRAM-1, RAR? and GSK3-? proteins by Western Blotting; assessing the activation of caspase-3 protein; clonal culture assay; immunophenotyping to CD11b and CD15; production and infections of lentivirus and assessment of morphological changes by Leishman stain. Results: Initially, referring to the possible increase of the cytotoxic effects of Ara-C by LIT in NB4 cells, it was found that LIT was not cytotoxic when used alone and did not increase the cytotoxic effects of Ara-C in this lineage. In relation to the increase of Ara-C activity in HL-60 cells, verified in previous studies, using the LIT as inhibitor of glycogen synthase kinase 3 (GSK3), it was verified na partial involvement of caspase-3 protein in AC+LIT association. Although the cells treated with Ara-C have shown significant decrease in protein expression levels of RAR?, increase in CD11b marker and decrease in CD15, these parameters were not altered when these associated LIT. It was also observed that silencing GSK3-? inhibited the increase of the cytotoxic effects of Ara-C by LIT, denoting the importance of this signaling pathway in the increase of the cytotoxic activity of Ara-C by LIT. The studies using the URS demonstrated significant cytotoxic effects in a concentration and time dependent manner, triggering many different cell death types, such as necrosis, apoptosis and late apoptosis after 4 hours of exposure. The URS also decreased the number of colonies, modulates autophagy and led the formation of vacuoles. The URS did not affect the cell differentiation markers CD11b and CD15, nor levels of PRAM-1 protein in NB4 cell line. The effects of URS for both cell lines were not cycle-dependent, once changes in cell cycle phases were not observed. URS did not alter the levels of GSK3-? protein, and did not increase the cytotoxic effects caused by Ara-C. Conclusions: It is suggested that the increase in cytotoxic effects of Ara-C depend on LIT inhibiting GSK3-?, thus stimulating further studies aimed at elucidating this action for future therapeutic targets in leucemias.Regarding the antileukemic effects of URS, this was significantly cytotoxic to the cell lines, possibly due to its modulation of the autophagic process and formation of vacuoles, effects that occurred independently of actions on the cell cycle and in GSK3-?, suggesting thus lower cytotoxic activity for normal tissues, that stimulates preclinical studies for possible application in antileukemic therapy.