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1.
Cell Biol Int ; 48(4): 521-540, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38263578

RESUMO

The ion channel two-pore channel 2 (TPC2), localised on the membranes of acidic organelles such as endo-lysosomes and melanosomes, has been shown to play a role in pathologies including cancer, and it is differently expressed in primary versus metastatic melanoma cells. Whether TPC2 plays a pro- or anti-oncogenic role in different tumour conditions is a relevant open question which we have explored in melanoma at different stages of tumour progression. The behaviour of primary melanoma cell line B16F0 and its metastatic subline B16F10 were compared in response to TPC2 modulation by silencing (by small interfering RNA), knock-out (by CRISPR/Cas9) and overexpression (by mCherry-TPC2 transfected plasmid). TPC2 silencing increased cell migration, epithelial-to-mesenchymal transition and autophagy in the metastatic samples, but abated them in the silenced primary ones. Interestingly, while TPC2 inactivation failed to affect markers of proliferation in both samples, it strongly enhanced the migratory behaviour of the metastatic cells, again suggesting that in the more aggressive phenotype TPC2 plays a specific antimetastatic role. In line with this, overexpression of TPC2 in B16F10 cells resulted in phenotype rescue, that is, a decrease in migratory ability, thus collectively resuming traits of the B16F0 primary cell line. Our research shows a novel role of TPC2 in melanoma cells that is intriguingly different in initial versus late stages of cancer progression.


Assuntos
Melanoma , Humanos , Melanoma/metabolismo , Canais de Dois Poros , Lisossomos/metabolismo , Linhagem Celular , Autofagia/fisiologia , Cálcio/metabolismo
2.
Environ Res ; 244: 117936, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38109963

RESUMO

The presence of plastic fragments in aquatic environments, particularly at the micro- and nano-scale, has become a significant global concern. However, current detection methods are limited in their ability to reveal the presence of such particles in liquid samples. In this study, we propose the use of a fluorescence lifetime analysis system for the detection of micro- and nanoplastics in water. This approach relies on the inherent endogenous fluorescence of plastic materials and involves the collection of single photons emitted by plastic fragments upon exposure to a pulsed laser beam. Briefly, a pulsed laser beam (repetition frequency = 40 MHz) shines onto a sample solution, and the emitted light is filtered, collected, and used to trace the time distributions of the photons with high temporal resolution. Finally, the fluorescence lifetime was measured using fitting procedures and a phasor analysis. Phasor analysis is a fit-free method that allows the measurement of the fluorescence lifetime of a sample without any assumptions or prior knowledge of the sample decay pattern. The developed instrument was tested using fluorescence references and validated using unlabelled micro- and nano-scale particles. Our system successfully detected polystyrene particles in water, achieving a remarkable sensitivity with a detection limit of 0.01 mg/mL, without the need for sample pre-treatment or visual inspection. Although further studies are necessary to enhance the detection limit of the technique and distinguish between different plastic materials, this proof-of-concept study suggests the potential of the fluorescence lifetime-based approach as a rapid, robust, and cost-effective method for early warning detection and identification of plastic contaminants in aquatic environments.


Assuntos
Microplásticos , Poluentes Químicos da Água , Microplásticos/análise , Água , Fluorescência , Poluentes Químicos da Água/análise , Poliestirenos/análise , Plásticos/análise
3.
Int J Mol Sci ; 21(22)2020 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-33212946

RESUMO

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.


Assuntos
Carcinoma Embrionário/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Neoplasias Testiculares/metabolismo , Carcinoma Embrionário/genética , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Fator de Crescimento de Hepatócito/genética , Humanos , Masculino , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Neoplasias Testiculares/genética
4.
Int J Mol Sci ; 20(18)2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31547344

RESUMO

Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca2+]i). The extremely complex calcium machinery includes plasma membrane bound channels as well as intracellular receptors distributed in distinct cytosolic compartments that act jointly to maintain a physiological [Ca2+]i, which is crucial for triggering many cellular mechanisms. Here, we first survey the overall notions related to intracellular Ca2+ mobilization and later highlight the involvement of this second messenger in crucial ECs functions with the aim at stimulating further investigation that link Ca2+ mobilization to ECs in health and disease.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Células Endoteliais/metabolismo , Animais , Canais de Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Células Endoteliais/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica
5.
J Cell Biochem ; 119(7): 5060-5071, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29637636

RESUMO

Caveolae are 50- to 100-nm cholesterol and glycosphingolipid-rich flask-shaped invaginations commonly observed in many terminally differentiated cells. These organelles have been described in many cell types and are particularly abundant in endothelial cells, where they have been involved in the regulation of certain signaling pathways. Specific scaffolding proteins termed caveolins, along with the more recently discovered members of the cavin family, represent the major protein components during caveolae biogenesis. In addition, multiple studies aimed to investigate the expression and the regulation of these proteins significantly contributed to elucidate the role of caveolae and caveolins in endothelial cell physiology and disease. The aim of this review is to survey recent evidence of the involvement of the caveolar network in endothelial cell biology and endothelial cell dysfunction.


Assuntos
Cavéolas/metabolismo , Endotélio/metabolismo , Animais , Caveolinas/metabolismo , Colesterol/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Transdução de Sinais/fisiologia
6.
Malar J ; 16(1): 366, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28899381

RESUMO

BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.


Assuntos
Antimaláricos/farmacologia , Carbolinas/farmacologia , NADP/análogos & derivados , Piperazinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Transdução de Sinais , Eritrócitos/parasitologia , Humanos , NADP/fisiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/fisiologia , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento , Esquizontes/fisiologia
7.
Proc Natl Acad Sci U S A ; 111(44): E4706-15, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25331892

RESUMO

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carbolinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Camundongos , Camundongos Knockout , NADP/análogos & derivados , NADP/antagonistas & inibidores , NADP/genética , NADP/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Piperazinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
8.
Int J Mol Sci ; 18(6)2017 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-28617309

RESUMO

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiating melanosphere-derived cells. Increased lipid uptake was found in melanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology.


Assuntos
Autofagia , Metabolismo dos Lipídeos , Melanoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Diferenciação Celular , Humanos , Melanoma/patologia , Células-Tronco Neoplásicas/patologia , PPAR gama/metabolismo , Fosforilação , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas
9.
J Biol Chem ; 290(9): 5470-83, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25568326

RESUMO

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-ß, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-ß expression.


Assuntos
Apoptose/genética , Poli I-C/genética , Receptores de Superfície Celular/genética , Transdução de Sinais/genética , Adjuvantes Imunológicos/metabolismo , Androgênios/metabolismo , Western Blotting , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferon beta/metabolismo , Masculino , Microscopia Confocal , Poli I-C/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Interferência de RNA , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
Biochim Biophys Acta ; 1851(7): 929-36, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25746012

RESUMO

c-Flip proteins are well-known apoptosis modulators. They generally contribute to tissue homeostasis maintenance by inhibiting death-receptor-mediated cell death. In the present manuscript, we show that c-Flip knock-out (KO) mouse embryonic fibroblasts (MEFs) kept in culture under starvation conditions gradually modify their phenotype and accumulate vacuoles, becoming progressively larger according to the duration of starvation. Large vacuoles are present in KO MEFs though not in WT MEFs, and are Oil Red-O positive, which indicates that they represent lipid droplets. Western blot experiments reveal that, unlike WT MEFs, KO MEFs express high levels of the lipogenic transcription factor PPAR-γ. Lipid droplet accumulation was found to be associated with endoplasmic reticulum (ER) stress activation and autophagic modulation valuated by means of BIP increase, LC3 lipidation and AMP-activated protein kinase (AMPK) phosphorylation, and p62 accumulation. Interestingly, XBP-1, an ER stress-induced lipogenic transcription factor, was found to preferentially localize in the nucleus rather than in the cytoplasm of KO MEFs. These data demonstrate that, upon starvation, c-Flip affects lipid accumulation, ER stress and autophagy, thereby pointing to an important role of c-Flip in the adaptive response and ER stress response programs under both normal and pathological conditions.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Estresse do Retículo Endoplasmático/genética , Fibroblastos/metabolismo , Metabolismo dos Lipídeos/genética , Animais , Células Cultivadas , Meios de Cultura Livres de Soro , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Técnicas de Silenciamento de Genes , Lipogênese/genética , Camundongos , PPAR gama/metabolismo , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-Box
12.
Proc Natl Acad Sci U S A ; 110(24): 9812-7, 2013 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-23716670

RESUMO

Toll-like receptor 3 (TLR3) is a key effector of the innate immune system against viruses. Activation of TLR3 exerts an antitumoral effect through a mechanism of action still poorly understood. Here we show that TLR3 activation by polyinosinic:polycytidylic acid induces up-regulation of microRNA-29b, -29c, -148b, and -152 in tumor-derived cell lines and primary tumors. In turn, these microRNAs induce reexpression of epigenetically silenced genes by targeting DNA methyltransferases. In DU145 and TRAMP-C1 prostate and MDA-MB-231 breast cancer cells, we demonstrated that polyinosinic:polycytidylic acid-mediated activation of TLR3 induces microRNAs targeting DNA methyltransferases, leading to demethylation and reexpression of the oncosuppressor retinoic acid receptor beta (RARß). As a result, cancer cells become sensitive to retinoic acid and undergo apoptosis both in vitro and in vivo. This study provides evidence of an antitumoral mechanism of action upon TLR3 activation and the biological rationale for a combined TLR3 agonist/retinoic acid treatment of prostate and breast cancer.


Assuntos
MicroRNAs/genética , Neoplasias/genética , Receptores do Ácido Retinoico/genética , Receptor 3 Toll-Like/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Poli I-C/farmacologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/prevenção & controle , Receptores do Ácido Retinoico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/metabolismo , Tretinoína/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Cell Mol Med ; 19(2): 327-39, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25444175

RESUMO

Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.


Assuntos
Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator Regulador 3 de Interferon/metabolismo , Próstata/efeitos dos fármacos , Próstata/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD
14.
Mediators Inflamm ; 2015: 417281, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26491226

RESUMO

Different stressful conditions such as hypoxia, nutrient deprivation, pH changes, or reduced vascularization, potentially able to act as growth-limiting factors for tumor cells, activate the unfolded protein response (UPR). UPR is therefore involved in tumor growth and adaptation to severe environments and is generally cytoprotective in cancer. The present review describes the molecular mechanisms underlying UPR and able to promote survival and proliferation in cancer. The critical role of UPR activation in tumor growth promotion is discussed in detail for a few paradigmatic tumors such as prostate cancer and melanoma.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Microambiente Tumoral/fisiologia , Animais , Humanos , Masculino , Melanoma/metabolismo , Neoplasias da Próstata/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
15.
Biol Reprod ; 90(3): 53, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24478388

RESUMO

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4(+)CD25(-)EGFP(-) T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4(+)FOXP3(-) T cells into functional CD4(+)FOXP3(+) Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Receptores Notch/fisiologia , Células de Sertoli/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Western Blotting , Antígenos CD4/biossíntese , Antígenos CD4/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Imunoprecipitação da Cromatina , Citometria de Fluxo , Fatores de Transcrição Forkhead/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Masculino , Proteínas de Membrana/genética , Camundongos , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , Receptores Notch/genética , Proteínas Serrate-Jagged , Supressão Genética , Transfecção , Fator de Crescimento Transformador beta/fisiologia
16.
Cells ; 13(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38474330

RESUMO

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Fenótipo , Proteômica , Células de Schwann/patologia , Fator de Crescimento Transformador beta/genética , Invasividade Neoplásica
17.
J Cell Mol Med ; 17(6): 713-22, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23551576

RESUMO

Toll-Like receptors (TLRs) are a family of evolutionary conserved transmembrane proteins that recognize highly conserved molecules in pathogens. TLR-expressing cells represent the first line of defence sensing pathogen invasion, triggering innate immune responses and subsequently priming antigen-specific adaptive immunity. In vitro and in vivo studies on experimental cancer models have shown both anti- and pro-tumoural activity of different TLRs in prostate cancer, indicating these receptors as potential targets for cancer therapy. In this review, we highlight the intriguing duplicity of TLR stimulation by pathogens: their protective role in cases of acute infections, and conversely their negative role in favouring hyperplasia and/or cancer onset, in cases of chronic infections. This review focuses on the role of TLRs in the pathophysiology of prostate infection and cancer by exploring the biological bases of the strict relation between TLRs and prostate cancer. In particular, we highlight the debated question of how reliable mutations or deregulated expression of TLRs are as novel diagnostic or prognostic tools for prostate cancer. So far, the anticancer activity of numerous TLR ligands has been evaluated in clinical trials only in organs other than the prostate. Here we review recent clinical trials based on the most promising TLR agonists in oncology, envisaging a potential application also in prostate cancer therapy.


Assuntos
Infecções Bacterianas/imunologia , Próstata/imunologia , Hiperplasia Prostática/imunologia , Neoplasias da Próstata/imunologia , Receptores Toll-Like/genética , Imunidade Adaptativa , Infecções Bacterianas/complicações , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/patologia , Biomarcadores/metabolismo , Ensaios Clínicos como Assunto , Expressão Gênica , Humanos , Imunidade Inata , Fatores Imunológicos/farmacologia , Masculino , Próstata/efeitos dos fármacos , Próstata/patologia , Hiperplasia Prostática/complicações , Hiperplasia Prostática/tratamento farmacológico , Hiperplasia Prostática/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Transdução de Sinais , Receptores Toll-Like/imunologia
18.
J Cell Biochem ; 114(8): 1843-51, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23463606

RESUMO

Caveolin-1 (CAV1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we investigated the involvement of CAV1 in endothelial cell (EC) functions and show that siRNA-induced CAV1 silencing in the human EC line EA.hy926 induces distinctive morphological changes, such as a marked increase in cell size and formation of stress fibers. Design-based stereology was employed in this work to make unbiased quantification of morphometric properties such as volume, length, and surface of CAV1 silenced versus control cells. In addition, we showed that downregulation of CAV1 affects cell cycle progression at G1/S phase transition most likely by perturbation of AKT signaling. With the aim to assess the contribution of CAV1 to typical biological processes of EC, we report here that CAV1 targeting affects cell migration and matrix metalloproteinases (MMPs) activity, and reduces angiogenesis in response to VEGF, in vitro. Taken together our data suggest that the proper expression of CAV1 is important not only for maintaining the appropriate morphology and size of ECs but it might represent a prospective molecular target for studying key biological mechanisms such as senescence and tumorigenesis.


Assuntos
Caveolina 1/biossíntese , Movimento Celular/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Linhagem Celular , Colagenases/metabolismo , Fase G1/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase S/fisiologia , Transdução de Sinais/fisiologia
19.
Blood ; 117(18): 4968-77, 2011 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-21364192

RESUMO

A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca(2+) release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca(2+) from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca(2+)-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca(2+) mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca(2+)-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.


Assuntos
Células Endoteliais/metabolismo , NADP/análogos & derivados , Receptores Histamínicos H1/metabolismo , Fator de von Willebrand/metabolismo , Sequência de Bases , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carbolinas/farmacologia , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Ácidos Heptanoicos/farmacologia , Histamina/farmacologia , Humanos , NADP/metabolismo , Antagonistas Nicotínicos/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , RNA Interferente Pequeno/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia
20.
Int J Mol Sci ; 14(6): 12090-106, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23743823

RESUMO

The role of autophagy is known to be highly complex and context-dependent, leading to both cancer suppression and progression in several tumors including melanoma, breast and prostate cancer. In the present review, recent advances in an understanding of the involvement of autophagy in prostate cancer treatment are described. The regulatory effects of androgens on prostate cancer cell autophagy are particularly discussed in order to highlight the effects of autophagy modulation during androgen deprivation. A critical evaluation of the studies examined in the present review suggests the attractive possibility of autophagy inhibition combined with hormonal therapy as a promising approach for prostate cancer treatment.


Assuntos
Androgênios/farmacologia , Androgênios/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Animais , Progressão da Doença , Humanos , Imunidade/efeitos dos fármacos , Masculino , Modelos Biológicos , Neoplasias da Próstata/imunologia
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