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1.
Cell Death Dis ; 12(7): 686, 2021 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-34238932

RESUMEN

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP-/- than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability.


Asunto(s)
Autofagia , Beclina-1/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Fibroblastos/metabolismo , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Fibroblastos/patología , Células HEK293 , Humanos , Ratones , Simulación del Acoplamiento Molecular , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Estabilidad Proteica , Ubiquitinación
2.
Front Cell Dev Biol ; 9: 629182, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33614661

RESUMEN

Oleic acid (OA) is a component of the olive oil. Beneficial health effects of olive oil are well-known, such as protection against liver steatosis and against some cancer types. In the present study, we focused on OA effects in hepatocellular carcinoma (HCC), investigating responses to OA treatment (50-300 µM) in HCC cell lines (Hep3B and Huh7.5) and in a healthy liver-derived human cell line (THLE-2). Upon OA administration higher lipid accumulation, perilipin-2 increase, and autophagy reduction were observed in HCC cells as compared to healthy cells. OA in the presence of 10% FBS significantly reduced viability of HCC cell lines at 300 µM through Alamar Blue staining evaluation, and reduced cyclin D1 expression in a dose-dependent manner while it was ineffective on healthy hepatocytes. Furthermore, OA increased cell death by about 30%, inducing apoptosis and necrosis in HCC cells but not in healthy hepatocytes at 300 µM dosage. Moreover, OA induced senescence in Hep3B, reduced P-ERK in both HCC cell lines and significantly inhibited the antiapoptotic proteins c-Flip and Bcl-2 in HCC cells but not in healthy hepatocytes. All these results led us to conclude that different cell death processes occur in these two HCC cell lines upon OA treatment. Furthermore, 300 µM OA significantly reduced the migration and invasion of both HCC cell lines, while it has no effects on healthy cells. Finally, we investigated autophagy role in OA-dependent effects by using the autophagy inducer torin-1. Combined OA/torin-1 treatment reduced lipid accumulation and cell death as compared to single OA treatment. We therefore concluded that OA effects in HCC cells lines are, at least, in part dependent on OA-induced autophagy reduction. In conclusion, we report for the first time an autophagy dependent relevant anti-cancer effect of OA in human hepatocellular carcinoma cell lines.

3.
Anticancer Res ; 40(10): 5379-5391, 2020 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-32988857

RESUMEN

BACKGROUND/AIM: Hypoxia-inducible factor 1 (HIF1) inhibitors have been proposed as therapeutic agents for several tumor types. HIF1α is induced by hypoxia and by pathogens in normoxia through toll-like receptors (TLRs). The TLR3 activator polyinosinic:polycytidylic acid [poly(I:C)] induces apoptosis in various types of cancer but not in the most aggressive breast cancer cell lines. We hypothesized that the failure of TLR3 stimulation to induce apoptosis in these cells might be due to an elevated HIF1α level and this link might be exploited. MATERIALS AND METHODS: Poly(I:C)-induced signaling pathway and expression of HIF1α and HIF1α targets were studied in MDA MB-231 and MCF-7 breast cancer cell lines by western blot. Flow cytometry was used for apoptotic responses and vasculogenic mimicry as bioassay. RESULTS: Poly(I:C) increased expression of HIF1α and its targets BCL2 apoptosis regulator and c-MYC. Moreover, using pharmacological or genetic HIF1 inhibition, reduction of poly(I:C)-induced expression of HIF1α was paralleled by lowering of c-MYC and increased sensitivity to poly(I:C)-induced apoptosis, demonstrating the crucial role of this factor. We provide the first evidence in breast cancer cells that TLR3 stimulation induces HIF1α-dependent vasculogenic mimicry. By using specific inhibitors, we identified a signaling cascade upstream of HIF1α induction. CONCLUSION: Combined treatment with poly(I:C) and HIF1 inhibitors deserves consideration as an effective strategy in breast cancer therapy.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Poli I-C/farmacología , Receptor Toll-Like 3/genética , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/agonistas
4.
Nutrients ; 11(4)2019 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-30979078

RESUMEN

: Autophagy plays a role in several physiological and pathological processes as it controls the turnover rate of cellular components and influences cellular homeostasis. The liver plays a central role in controlling organisms' metabolism, regulating glucose storage, plasma proteins and bile synthesis and the removal of toxic substances. Liver functions are particularly sensitive to autophagy modulation. In this review we summarize studies investigating how autophagy influences the hepatic metabolism, focusing on fat accumulation and lipids turnover. We also describe how autophagy affects bile production and the scavenger function within the complex homeostasis of the liver. We underline the role of hepatic autophagy in counteracting the metabolic syndrome and the associated cardiovascular risk. Finally, we highlight recent reports demonstrating how the autophagy occurring within the liver may affect skeletal muscle homeostasis as well as different extrahepatic solid tumors, such as melanoma.


Asunto(s)
Autofagia/fisiología , Células Epiteliales/fisiología , Homeostasis/fisiología , Hígado/citología , Conductos Biliares/citología , Muerte Celular , Dieta , Dislipidemias , Células Endoteliales/fisiología , Ingestión de Energía , Hepatocitos/fisiología , Humanos , Macrófagos del Hígado/fisiología , Metabolismo de los Lípidos/fisiología , Lipólisis , Melanoma , Músculo Esquelético/metabolismo , Neoplasias , Estrés Oxidativo
5.
Oncotarget ; 9(56): 30905-30918, 2018 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-30112117

RESUMEN

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.

6.
Int J Mol Sci ; 18(6)2017 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-28617309

RESUMEN

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.


Asunto(s)
Autofagia , Metabolismo de los Lípidos , Melanoma/metabolismo , Células Madre Neoplásicas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Diferenciación Celular , Humanos , Melanoma/patología , Células Madre Neoplásicas/patología , PPAR gamma/metabolismo , Fosforilación , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales Cultivadas
7.
Oxid Med Cell Longev ; 2017: 4629495, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29379583

RESUMEN

GSK-3 is a ubiquitously expressed serine/threonine kinase existing as GSK-3α and GSK-3ß isoforms, both active under basal conditions and inactivated upon phosphorylation by different upstream kinases. Initially discovered as a regulator of glycogen synthesis, GSK-3 is also involved in several signaling pathways controlling many different key functions. Here, we discuss recent advances regarding (i) GSK-3 structure, function, regulation, and involvement in several cancers, including hepatocarcinoma, cholangiocarcinoma, breast cancer, prostate cancer, leukemia, and melanoma (active GSK-3 has been shown to induce apoptosis in some cases or inhibit apoptosis in other cases and to induce cancer progression or inhibit tumor cell proliferation, suggesting that different GSK-3 modulators may address different specific targets); (ii) GSK-3 involvement in autophagy modulation, reviewing signaling pathways involved in neurodegenerative and liver diseases; (iii) GSK-3 role in oxidative stress and autophagic cell death, focusing on liver injury; (iv) GSK-3 as a possible therapeutic target of natural substances and synthetic inhibitors in many diseases; and (v) GSK-3 role as modulator of mammalian aging, related to metabolic alterations characterizing senescent cells and age-related diseases. Studies summarized here underline the GSK-3 multifaceted role and indicate such kinase as a molecular target in different pathologies, including diseases associated with autophagy dysregulation.


Asunto(s)
Autofagia , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Hepatopatías/enzimología , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Enfermedades Neurodegenerativas/enzimología , Animales , Apoptosis , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Humanos , Hepatopatías/genética , Hepatopatías/patología , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Estrés Oxidativo
8.
Infect Immun ; 84(11): 3105-3113, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27600504

RESUMEN

Adherent/invasive Escherichia coli (AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenic E. coli (ExPEC), neonatal meningitis-associated E. coli (NMEC), and uropathogenic E. coli (UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers. In silico analysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Our in vitro data support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract.


Asunto(s)
Adhesión Bacteriana/fisiología , Células Epiteliales/microbiología , Escherichia coli/patogenicidad , Próstata/citología , Biopelículas/crecimiento & desarrollo , Línea Celular , Enfermedad de Crohn/microbiología , Células Epiteliales/metabolismo , Escherichia coli/fisiología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Humanos , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fenotipo , Filogenia , Virulencia , Factores de Virulencia/metabolismo
9.
Cell Signal ; 28(9): 1262-1269, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27267061

RESUMEN

Cellular-Flice-like inhibitory protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the endoplasmic reticulum (ER) and that c-FLIP-deficient mouse embryonic fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP-/- MEFs. Surprisingly, c-FLIP-/- MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP-/- cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP-/- MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP-/- MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP-/- cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response.


Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Estrés del Retículo Endoplásmico , Animales , Apoptosis , Autofagia , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/deficiencia , Embrión de Mamíferos/citología , Activación Enzimática , Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , eIF-2 Quinasa/metabolismo
10.
New Microbiol ; 39(2): 146-9, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27196555

RESUMEN

RWPE-1 normal prostate cells were tested as an experimental model for adhesion/invasion assays by genotypically and phenotypically characterized community uropathogenic strains of Escherichia coli (UPEC), a frequent cause of urinary tract infections (UTIs) and significant etiologic agent also in bacterial prostatitis. Adhesive ability and strong biofilm production was significantly associated with the bacterial invasive phenotype. Invasive strains derived mainly from male and pediatric patients. This study suggests that such a cell model could usefully integrate other available methods of urovirulence analysis, to deepen knowledge on the bacterial interaction with host cells.


Asunto(s)
Infecciones por Escherichia coli/microbiología , Próstata/citología , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/fisiología , Adolescente , Adulto , Adhesión Bacteriana/fisiología , Línea Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Escherichia coli Uropatógena/clasificación , Adulto Joven
11.
Sci Rep ; 6: 18925, 2016 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-26733361

RESUMEN

A novel transduction pathway for the powerful angiogenic factor VEGF has been recently shown in endothelial cells to operate through NAADP-controlled intracellular release of Ca(2+). In the present report the possible involvement of NAADP-controlled Ca(2+) signaling in tumor vascularization, growth and metastatic dissemination was investigated in a murine model of VEGF-secreting melanoma. Mice implanted with B16 melanoma cells were treated with NAADP inhibitor Ned-19 every second day for 4 weeks and tumor growth, vascularization and metastatization were evaluated. Control specimens developed well vascularized tumors and lung metastases, whereas in Ned-19-treated mice tumor growth and vascularization as well as lung metastases were strongly inhibited. In vitro experiments showed that Ned-19 treatment controls the growth of B16 cells in vitro, their migratory ability, adhesive properties and VEGFR2 expression, indicating NAADP involvement in intercellular autocrine signaling. To this regard, Ca(2+) imaging experiments showed that the response of B16 cells to VEGF stimulation is NAADP-dependent. The whole of these observations indicate that NAADP-controlled Ca(2+) signaling can be relevant not only for neoangiogenesis but also for direct control of tumor cells.


Asunto(s)
Señalización del Calcio , Melanoma/metabolismo , Melanoma/patología , NADP/análogos & derivados , Neovascularización Patológica/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Carbolinas/farmacocinética , Carbolinas/farmacología , Carbolinas/toxicidad , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Masculino , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma Experimental , Ratones , NADP/metabolismo , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Piperazinas/farmacocinética , Piperazinas/farmacología , Piperazinas/toxicidad , Carga Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
13.
Mediators Inflamm ; 2015: 417281, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26491226

RESUMEN

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.


Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Microambiente Tumoral/fisiología , Animales , Humanos , Masculino , Melanoma/metabolismo , Neoplasias de la Próstata/metabolismo , Respuesta de Proteína Desplegada/fisiología
14.
Biomed Res Int ; 2015: 965271, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26146638

RESUMEN

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca(2+) signaling, antagonizing IP3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by "scratch assay," was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca(2+) release specifically regulates distinct Angs-mediated angiogenic steps.


Asunto(s)
Angiotensina II/metabolismo , Señalización del Calcio/genética , ADP-Ribosa Cíclica/genética , Neovascularización Fisiológica/genética , Angiopoyetinas/genética , Angiopoyetinas/metabolismo , Angiotensina II/genética , Calcio/metabolismo , Proliferación Celular/genética , ADP-Ribosa Cíclica/biosíntesis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Receptor TIE-2/genética , Receptor TIE-2/metabolismo
15.
Biochim Biophys Acta ; 1851(7): 929-36, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25746012

RESUMEN

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.


Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Estrés del Retículo Endoplásmico/genética , Fibroblastos/metabolismo , Metabolismo de los Lípidos/genética , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Técnicas de Silenciamiento del Gen , Lipogénesis/genética , Ratones , PPAR gamma/metabolismo , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box
16.
J Biol Chem ; 290(9): 5470-83, 2015 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-25568326

RESUMEN

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.


Asunto(s)
Apoptosis/genética , Poli I-C/genética , Receptores de Superficie Celular/genética , Transducción de Señal/genética , Adyuvantes Inmunológicos/metabolismo , Andrógenos/metabolismo , Western Blotting , Línea Celular Tumoral , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1 , Interferón beta/genética , Interferón beta/metabolismo , Masculino , Microscopía Confocal , Poli I-C/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Interferencia de ARN , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Transfección , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
17.
J Mater Chem B ; 3(37): 7408-7416, 2015 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-32262767

RESUMEN

Polyinosinic-polycytidylic acid (poly(I:C)) is a synthetic double-stranded RNA (dsRNA) analog able to induce apoptosis in different cancer cells by the activation of toll-like receptor 3 (TLR3) and cytosolic helicases, retinoic acid inducible gene I (RIG-I) like receptors. In this work, we have synthesized and thoroughly characterized a core-shell liposome-silica hybrid (LSH) nanoparticle (NP) made of a silica core surrounded by a multicomponent cationic lipid bilayer. In view of in vivo applications, a variant with polyethyleneglycol (PEG) grafted onto the lipid surface was also synthesized. Poly(I:C)-loaded LSH NPs were characterized and optimized in terms of their chemical-physical properties by using dynamic light scattering (DLS), micro-electrophoresis and transmission electron microscopy (TEM). The ability of this new technology to kill cancer cells was validated in PC3 prostate cancer and MCF7 breast cancer cells by MTT proliferation assay, flow cytometry and fluorescence confocal microscopy. We found that negatively charged poly(I:C)-loaded LSH NPs are more efficient than their liposome counterpart in eliminating cancer cells, thus representing excellent candidates for both in vitro and in vivo drug delivery applications.

18.
J Cell Mol Med ; 19(2): 327-39, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25444175

RESUMEN

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.


Asunto(s)
Andrógenos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor 3 Regulador del Interferón/metabolismo , Próstata/efectos de los fármacos , Próstata/metabolismo , Animales , Apoptosis/fisiología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD
19.
Proc Natl Acad Sci U S A ; 111(44): E4706-15, 2014 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-25331892

RESUMEN

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.


Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Canales de Calcio/genética , Señalización del Calcio/efectos de los fármacos , Carbolinas/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Ratones , Ratones Noqueados , NADP/análogos & derivados , NADP/antagonistas & inhibidores , NADP/genética , NADP/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Piperazinas/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética
20.
Forensic Sci Int Genet ; 11: 137-43, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24727431

RESUMEN

The correct identification of the biological samples under analysis is crucial in forensic investigation in that it represents the pivotal issue attesting that the resulting genetic profiles are fully reliable in terms of weight of the evidence. The study reported herein shows that "touch DNA" secondary transfer is indeed possible from person to person and, in turn, from person to object depending on the specific sebaceous or non-sebaceous skin area previously touched. In addition, we demonstrate the presence of fragmented single stranded DNA specifically immunodetected in the vast majority of cells forming the sebaceous gland but not in the epidermis layers, strongly indicating that sebaceous fluid represents an important vector responsible for DNA transfer. In view of our results, forensic investigations need to take into account that the propensity to leave behind genetic material through contact could depend from the individual ability to shed sebaceous fluid on the skin surface.


Asunto(s)
Dermatoglifia del ADN , Piel/metabolismo , Humanos
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