Navegando por Palavras-chave "Cytoskeleton"
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- ItemSomente MetadadadosAnálise molecular do tráfego intracelular do canal para prótons sensível à voltagem por microscopia de fluorescência(Universidade Federal de São Paulo (UNIFESP), 2021) Silva, Luisa Ribeiro [UNIFESP]; Miranda Filho, Manoel de Arcisio [UNIFESP]; Universidade Federal de São PauloVoltage-gated proton channel presents a physiological role of great importance to phagocytosis and sub sequential death of microorganisms in phagocytic cells, such as neutrophils and microglia. Recently various studies described the contribution of this channel in a higher malignity of tumors and its proliferation. Additionally, HV1 channels have been related to augmented tissue damage in cerebral stroke. Even though these pathological processes have been associated with a higher superficial expression of HV1 channels, little is known about how their abundance in the plasma membrane is regulated. Thus, in this work we have studied the molecular mechanisms of HV1 channel’s intracellular trafficking, and the relevance of cytoskeleton to its transport and function. Through advanced microscopy techniques, we showed that when overexpressed in a heterologous expression system, CHO-K1 cells, the channels are present diffuse in the plasma membrane and aggregated in vesicles in the region close to the plasma membrane. The movement of these vesicles containing HV1 was characterized for the first time in literature, as well as their fusion to the plasma membrane through kiss-and-run and/or kiss-and-linger mechanisms. Furthermore, high-resolution microscopy analysis of HV1 intracellular localization through co-transfection with 8 different intracellular organelles markers fused with EGFP resulted in a co-localization with caveolae, secretory pathway, late endosome, and recycling endosome. However, only the association with the recycling endosome was evident after dynamic co-localization analysis. The functional relationship between cytoskeleton and HV1 channels was analyzed using disruptive agents of microtubules and actin. Actin depolymerization resulted in a reduction of the density of activation and deactivation currents and diminished time to half peak of activation currents. The same effects were observed with microtubule depolymerization along with a higher activation voltage. On the other hand, cytoskeleton disruption did not arise alterations in the superficial expression and mobility of the channel, except for a lower diffusion coefficient with the loss of microtubules. Overall, these results suggests that HV1 channels are associated with recycling endosomes and are functionally modulated by both components of the cytoskeleton.
- ItemSomente MetadadadosAvaliação Da Atividade Do Amblyomin-X Na Regulação Da Adesão E Migração De Células Endoteliais E Tumorais São Paulo,(Universidade Federal de São Paulo (UNIFESP), 2017-02-24) Schmidt, Mariana Costa Braga [UNIFESP]; Tavassi, Ana Marisa Chudzinski [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Amblyomin-X is a Kunitz-type protease inhibitor that promotes phospholipids-dependent inhibition of FXa, or inhibits the activation of FX through TF/FVlla complex. This recombinant protein shows similarity with Kunitz domain of TFPI-1 and -2, that are endogenous regulators of coagulation process and has anti-tumor activity. On previous studies, Amblyomin-X showed anti-tumor effects, besides regulation of development and progression tumor process, like coagulation and angiogenesis. Furthermore, this molecule possesses cytotoxic activity associated to proteasome, endoplasmic reticulum stress, mitochondria dysfunction and caspase activation. However, the same didn't occur on normal cells like fibroblast, suggesting that this protease has selective cytotoxic activity on tumor cells. Although the advances were made in pro-apoptotic signaling on tumor cells, it is not yet elucidated Amblyomin-X action on pericelular proteolysis and extracellular matrix remodeling, important process on progression and metastasis tumor. After treatment of human umbilical vein endothelial cells (HUVECs) and melanoma (SK-MEL-28) and pancreas adenocarcinoma (MIA PaCa'~2) tumor cells with Amblyomin-X in an isolated or a conjunct way with inhibitors antiuPAR, Exo 1 (exocytosis) ou HPI-4 (dynein), we analyzed cell viability (MTT), quantified uPA and PAI-1 liberation (ELlSA), cytoskeleton (faloidin), migration (IN Cell), MMPs liberation (zymography) and uPAR, RhoA and Rac 1 expression (Western Blot). The results showed that Amblyomin-X has not cytotoxic effects ou induce cytoskeleton modification on HUVECs, but intensify interaction between uPAR and co-receptors, increasing adhesion and reducing migration of these cells. Though, tumor cells exhibit viability depletion, cytoskeleton and migration regulation by Amblyomin-X by different forms, decreasing Rho GTPase expression or reducing pericelular proteases and MMPs liberation. Besides that, Amblyomin-X reversed alterations promoted by uPAR antibody or exocytosis inhibition, which suggests a common pathway between Amblyomin-X and uPAR. Considering this data, we can affirm that Amblyomin-X has selectivity cytotoxic activity on tumor cells and regulate different pathways on tumor and endothelial cells that inhibits cell migration. Differently, bortezomib, an antitumor drug used in distinct cancer treatments, has harmful action on tumor and endothelial cells, which generates side effects discussed on literature. Therefore, the results showed on this work provides significant information about cytotoxic effects and regulation of migration activity by Amblyomin-X, which are important process to reduce growth and metastasis tumor, and give expectations to new studies in this subject.
- ItemSomente MetadadadosFibroblast and prostate tumor cell cross-talk: Fibroblast differentiation, TGF-beta, and extracellular matrix down-regulation(Elsevier B.V., 2010-11-15) Coulson-Thomas, Vivien Jane [UNIFESP]; Gesteira, Tarsis F.; Coulson-Thomas, Yvette May [UNIFESP]; Vicente, Carolina M.; Tersariol, Ivarne L. S.; Nader, Helena B.; Toma, Leny; Universidade Federal de São Paulo (UNIFESP)Growth and survival of tumors at a site of metastasis involve interactions with stromal cells in the surrounding environment. Stromal cells aid tumor cell growth by producing cytokines as well as by modifying the environment surrounding the tumor through modulation of the extracellular matrix (ECM). Small leucine-rich proteoglycans (SLRPs) are biologically active components of the ECM which can be altered in the stroma surrounding tumors. the influence tumor cells have on stromal cells has been well elucidated. However, little is understood about the effect metastatic cancer cells have on the cell biology and behavior of the local stromal cells. Our data reveal a significant downregulation in the expression of ECM components such as collagens I, II, III, and IV, and the SLRPs, decorin, biglycan, lumican, and fibromodulin in stromal cells when grown in the presence of two metastatic prostate cancer cell lines PO and DU145. Interestingly, TGF-beta down-regulation was observed in stromal cells, as well as actin depolymerization and increased vimentin and alpha 5 beta 1 integrin expression. MT1-MMP expression was upregulated and localized in stromal cell protrusions which extended into the ECM. Moreover, enhanced stromal cell migration was observed after crosstalk with metastatic prostate tumor cells. Xenografting metastatic prostate cancer cells together with activated stromal cells led to increased tumorigenicity of the prostate cancer cells. Our findings suggest that metastatic prostate cancer cells create a metastatic niche by altering the phenotype of local stromal cells, leading to changes in the ECM. (C) 2010 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosLumican expression, localization and antitumor activity in prostate cancer(Elsevier B.V., 2013-04-15) Coulson-Thomas, Vivien Jane [UNIFESP]; Coulson-Thomas, Yvette May [UNIFESP]; Gesteira, Tarsis Ferreira [UNIFESP]; Paula, Claudia Alessandra Andrade de [UNIFESP]; Carneiro, Celia Regina Whitaker [UNIFESP]; Ortiz, Valdemar [UNIFESP]; Toma, Leny [UNIFESP]; Kao, Winston W. Y.; Nader, Helena Bonciani [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ CincinnatiThe stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. the role of lumican in cancer varies according to the type of tumor. in this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. the increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. in vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin beta 1 and MT1-MMP, and invadopodia detected by disruption of alpha-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. in conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging. (C) 2013 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosThermoprotective effect of insulin-like growth factor 1 on in vitro matured bovine oocyte exposed to heat shock(Elsevier Science Inc, 2016) Rodrigues, Thais Alves [UNIFESP]; Ispada, Jessica [UNIFESP]; Risolia, Pedro Henrique Bugallo; Rodrigues, Mariana Teixeira [UNIFESP]; Lima, Rafaela S.; Assumpcao, Mayra Elena Ortiz D'Avila; Visintin, José Antônio; Paula-Lopes, Fabiola Freitas [UNIFESP]The role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 degrees C for 22 hours) and heat shock (41 degrees C for 14 hours followed by 38.5 degrees C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 degrees C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNEL-positive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration. (C) 2016 Elsevier Inc. All rights reserved.