RESUMEN

In the original publication [...].

RESUMEN

Mesoglycan is a fibrinolytic compound but recently promising pro-healing effects in skin wound repair have been reported. Previously, we have showed that mesoglycan activates human keratinocytes, fibroblasts and endothelial cells and induces the secretion of microvesicles (EVs), particularly exosomes, from keratinocytes. These EVs may contribute to wound healing since they further activate cells generating an autocrine loop with a positive feedback. In this work, EVs isolated from keratinocytes, treated with mesoglycan, have been tested on human fibroblasts and endothelial cells. The in vitro investigation has been carried out through Wound-Healing/invasion assays to analyze cell motility and assess the differentiation process. Then, the formation of capillary-like structures by human endothelial cells has been performed to evaluate in vitro angiogenesis. We found that EVs secreted from keratinocytes treated with mesoglycan promote fibroblasts and endothelial cells migration and invasion. Furthermore, these receiving cells acquire a mesenchymal phenotype. Additionally, the angiogenesis appears strongly enhanced in presence of this kind of EVs. In conclusion, we show that EVs deriving from keratinocytes trigger a paracrine positive feedback able to further amplify the effects of mesoglycan. This mechanism adds up to the autocrine loop previously reported and culminates with the activation of fibroblasts and endothelial cells. Particularly, this activation is amplified by the action of growth factors as FGF-2 (Fibroblast Growth Factor-2) for the fibroblasts and by VEGF (Vascular Endothelial Growth Factor) for the endothelial cells.

Asunto(s)

Células Endoteliales/efectos de los fármacos , Exosomas/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Glicosaminoglicanos/farmacología , Queratinocitos/citología , Línea Celular , Movimiento Celular/efectos de los fármacos , Células Endoteliales/fisiología , Fibroblastos/fisiología , Humanos , Piel , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

We have recently demonstrated that mesoglycan, a fibrinolytic compound, may be a promising pro-healing drug for skin wound repair. We showed that mesoglycan induces migration, invasion, early differentiation, and translocation to the membrane of keratinocytes, as well as the secretion of annexin A1 (ANXA1), further involved in keratinocytes activation. These events are triggered by the syndecan-4 (SDC4)/PKCα pathway. SDC4 also participates to the formation and secretion of microvesicles (EVs) which may contribute to wound healing. EVs were isolated from HaCaT cells, as human immortalized keratinocytes, and then characterised by Western blotting, Field Emission-Scanning Electron Microscopy, and Dynamic Light Scattering. Their autocrine effects were investigated by Wound-Healing/invasion assays and confocal microscopy to analyse cell motility and differentiation, respectively. Here, we found that the mesoglycan increased the release of EVs which amplify its same effects. ANXA1 contained in the microvesicles is able to promote keratinocytes motility and differentiation by acting on Formyl Peptide Receptors (FPRs). Thus, the extracellular form of ANXA1 may be considered as a link to intensify the effects of mesoglycan. In this study, for the first time, we have identified an interesting autocrine loop ANXA1/EVs/FPRs in human keratinocytes, induced by mesoglycan.

Asunto(s)

Anexina A1/fisiología , Vesículas Extracelulares/metabolismo , Glicosaminoglicanos/metabolismo , Queratinocitos/metabolismo , Receptores de Formil Péptido/metabolismo , Piel/metabolismo , Comunicación Autocrina , Diferenciación Celular , Línea Celular , Movimiento Celular , Humanos , Queratinocitos/citología , Piel/citología

RESUMEN

Wound healing is a dynamic process comprising multiple events, such as inflammation, re-epithelialization, and tissue remodeling. Re-epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re-epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non-healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re-epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan-4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new pro-healing drug for skin wound care.

Asunto(s)

Anexina A1/metabolismo , Glicosaminoglicanos/metabolismo , Queratinocitos/metabolismo , Proteínas S100/metabolismo , Sindecano-4/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Humanos , Repitelización/fisiología , Piel/metabolismo , Cicatrización de Heridas/fisiología

RESUMEN

Pancreatic Cancer (PC) is one of the most aggressive malignancies worldwide. As annexin A1 (ANXA1) is implicated in the establishment of tumour metastasis, the role of the protein in PC progression as a component of extracellular vesicles (EVs) has been investigated. EVs were isolated from wild type (WT) and ANXA1 knock-out (KO) PC cells and then characterised by multiple approaches including Western blotting, Field Emission-Scanning Electron Microscopy, and Dynamic Light Scattering. The effects of ANXA1 on tumour aggressiveness were investigated by Wound-Healing and invasion assays and microscopic analysis of the Epithelial to Mesenchymal Transition (EMT). The role of ANXA1 on angiogenesis was also examined in endothelial cells, using similar approaches. We found that WT cells released more EVs enriched in exosomes than those from cells lacking ANXA1. Notably, ANXA1 KO cells recovered their metastatic potential only when treated by WT EVs as they underwent EMT and a significant increase of motility. Similarly, human umbilical vein endothelial cells (HUVEC) migrated and invaded more rapidly when treated by WT EVs whereas ANXA1 KO EVs weakly induced angiogenesis. This study suggests that EVs-related ANXA1 is able to promote cell migration, invasion, and angiogenesis, confirming the relevance of this protein in PC progression.

Asunto(s)

Anexina A1/metabolismo , Progresión de la Enfermedad , Vesículas Extracelulares/metabolismo , Modelos Biológicos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Línea Celular Tumoral , Movimiento Celular , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal , Técnicas de Inactivación de Genes , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Invasividad Neoplásica , Fenotipo

RESUMEN

Annexin A1 (ANXA1) is a Ca2+-binding protein that is involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. Our previous studies showed that ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process appeared affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells finding that the modification in miRNA expression suggests the significant involvement of ANXA1 in PC development. In this study, we focused on miR-196a which appeared down modulated in absence of ANXA1. This miRNA is a well known oncogenic factor in several tumour models and it is able to trigger the agents of the epithelial to mesenchymal transition (EMT), like ANXA1. Our results show that the reintroduction in ANXA1 KO cells of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate cytoskeletal dynamics and other protein functions promoting PC cell migration and invasion. This work describes the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a. These results could lead to further information on ANXA1 intracellular role in PC, explaining other aspects that are apart from its tumorigenic behaviour.

Asunto(s)

Anexina A1/genética , MicroARNs/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Edición Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , MicroARNs/genética , Invasividad Neoplásica , Metástasis de la Neoplasia

RESUMEN

Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.

Asunto(s)

Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Queratinocitos/citología , Piel/citología , Movimiento Celular/fisiología , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos

RESUMEN

Glycosaminoglycans are polysaccharides of the extracellular matrix supporting skin wound closure. Mesoglycan is a mixture of glycosaminoglycans such as chondroitin-, dermatan-, heparan-sulfate and heparin and is the main component of Prisma® Skin, a pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. Here, we show the in vitro effects of this device in the new vessels formation by endothelial cells, since angiogenesis represents a key moment in wound healing. We found a strong increase of migration and invasion rates of these cells treated with mesoglycan and Prisma® Skin which mediate the activation of the pathway triggered by CD44 receptor. Furthermore, endothelial cells form longer capillary-like structures with a great number of branches, in the presence of the same treatments. Thus, the device, thanks to the mesoglycan, leads the cells to the Endothelial-to-Mesenchymal Transition, suggesting the switch to a fibroblast-like phenotype, as shown by immunofluorescence assays. Finally, we found that mesoglycan and Prisma® Skin inhibit inflammatory reactions such as nitric oxide secretion and NF-κB nuclear translocation in endothelial cells and Tumor Necrosis Factor-α production by macrophages. In conclusion, based on our data, we suggest that Prisma® Skin may be able to accelerate angiogenesis in skin wound healing, and regulate inflammation avoiding chronic, thus pathological, responses.

Asunto(s)

Transición Epitelial-Mesenquimal/efectos de los fármacos , Glicosaminoglicanos/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Piel/irrigación sanguínea , Cicatrización de Heridas/efectos de los fármacos , Movimiento Celular , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Receptores de Hialuranos/metabolismo , Técnicas In Vitro , Preparaciones Farmacéuticas

RESUMEN

BACKGROUND: Deflazacort is a synthetic corticosteroid characterized by a favourable pharmacokinetic profile, peculiar pharmacodynamic properties and a good safety profile. However, to the best of our knowledge, no dose-conversion table based on direct comparison of relative potencies between deflazacort and other main corticosteroids is currently available in scientific literature. MAIN BODY: This paper, while reporting a brief review of deflazacort pharmacological properties, its efficacy and tolerability in different clinical areas, has been designed with the specific aim of providing a new dose-conversion table of corticosteroids, including for the first time also deflazacort. SHORT CONCLUSION: We suggest that this new conversion table could be a useful tool for physicians who need to select the appropriate dose of deflazacort in their clinical practice.

Asunto(s)

Corticoesteroides/administración & dosificación , Antiinflamatorios/administración & dosificación , Pregnenodionas/administración & dosificación , Corticoesteroides/farmacocinética , Corticoesteroides/farmacología , Antiinflamatorios/farmacocinética , Antiinflamatorios/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Pregnenodionas/farmacocinética , Pregnenodionas/farmacología , Equivalencia Terapéutica , Índice Terapéutico

RESUMEN

Annexin A1 (ANXA1) is a Ca2+-binding protein overexpressed in the invasive stages of prostate cancer (PCa) development; however, its role in this tumor metastatization is largely unknown. Moreover, hypoxic conditions in solid tumors have been related to poor prognosis in PCa patients. We have previously demonstrated that ANXA1 is implicated in the acquisition of chemo-resistant features in DU145 PCa cells conferring them a mesenchymal/metastatic phenotype. In this study, we have investigated the mechanisms by which ANXA1 regulates metastatic behavior in LNCaP, DU145 and PC3 cells exposed to hypoxia. ANXA1 was differentially expressed by PCa cell lines in normoxia whereas hypoxic stimuli resulted in a significant increase of protein expression. Additionally, in low oxygen conditions ANXA1 was extensively secreted out-side the cells where its binding to formyl peptide receptors (FPRs) induced cell invasion. Loss and gain of function experiments performed by using the RNA interfering siANXA1 and an ANXA1 over-expressing plasmid (MF-ANXA1), also confirmed the leading role of the protein in modulating LNCaP, DU145 and PC3 cell invasiveness. Finally, ANXA1 played a crucial role in the regulation of cytoskeletal dynamics underlying metastatization process, such as the loss of adhesion molecules and the occurrence of the epithelial to mesenchymal transition (EMT). ANXA1 expression increased inversely to epithelial markers such as E-cadherin and cytokeratins 8 and 18 (CKs) and proportionally to mesenchymal ones such as vimentin, ezrin and moesin. Our results indicated that ANXA1 may be a key mediator of hypoxia-related metastasis-associated processes in PCa.

Asunto(s)

Anexina A1/genética , Movimiento Celular/genética , Invasividad Neoplásica/genética , Neoplasias de la Próstata/genética , Antígenos CD , Cadherinas/genética , Hipoxia de la Célula/genética , Línea Celular Tumoral , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Queratina-18/genética , Queratina-8/genética , Masculino , Invasividad Neoplásica/patología , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética

RESUMEN

Annexin A1 (ANXA1) is a Ca(2+)-binding protein over-expressed in pancreatic cancer (PC). We recently reported that extracellular ANXA1 mediates PC cell motility acting on Formyl Peptide Receptors (FPRs). Here, we describe other mechanisms by which intracellular ANXA1 could mediate PC progression. We obtained ANXA1 Knock-Out (KO) MIA PaCa-2 cells using the CRISPR/Cas9 genome editing technology. LC-MS/MS analysis showed altered expression of several proteins involved in cytoskeletal organization. As a result, ANXA1 KO MIA PaCa-2 partially lost their migratory and invasive capabilities with a mechanism that appeared independent of FPRs. The acquisition of a less aggressive phenotype has been further investigated in vivo. Wild type (WT), PGS (scrambled) and ANXA1 KO MIA PaCa-2 cells were engrafted orthotopically in SCID mice. No differences were found about PC primary mass, conversely liver metastatization appeared particularly reduced in ANXA1 KO MIA PaCa-2 engrafted mice. In summary, we show that intracellular ANXA1 is able to preserve the cytoskeleton integrity and to maintain a malignant phenotype in vitro. The protein has a relevant role in the metastatization process in vivo, as such it appears attractive and suitable as prognostic and therapeutic marker in PC progression.

Asunto(s)

Anexina A1/metabolismo , Metástasis de la Neoplasia/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Receptores de Formil Péptido/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Femenino , Humanos , Ratones , Ratones SCID , Fenotipo

RESUMEN

In this study, we have characterized the role of annexin A1 (ANXA1) in the acquisition and maintenance of stem-like/aggressive features in prostate cancer (PCa) cells comparing zoledronic acid (ZA)-resistant DU145R80 with their parental DU145 cells. ANXA1 is over-expressed in DU145R80 cells and its down-regulation abolishes their resistance to ZA. Moreover, ANXA1 induces DU145 and DU145R80 invasiveness acting through formyl peptide receptors (FPRs). Also, ANXA1 knockdown is able to inhibit epithelial to mesenchymal transition (EMT) and to reduce focal adhesion kinase (FAK) and metalloproteases (MMP)-2/9 expression in PCa cells. DU145R80 show a cancer stem cell (CSC)-like signature with a high expression of CSC markers including CD44, CD133, NANOG, Snail, Oct4 and ALDH7A1 and CSC-related genes as STAT3. Interestingly, ANXA1 knockdown induces these cells to revert from a putative prostate CSC to a more differentiated phenotype resembling DU145 PCa cell signature. Similar results are obtained concerning some drug resistance-related genes such as ATP Binding Cassette G2 (ABCG2) and Lung Resistant Protein (LRP). Our study provides new insights on the role of ANXA1 protein in PCa onset and progression.

Asunto(s)

Anexina A1/metabolismo , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Difosfonatos/farmacología , Resistencia a Antineoplásicos , Imidazoles/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Anexina A1/genética , Biomarcadores de Tumor/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Transfección , Ácido Zoledrónico

RESUMEN

In this paper, for the first time, hydrogels containing Annexin A1 N-terminal derived peptide, Ac2-26, as a novel dressing were successfully developed for dermal wound repair application. High mannuronic (M) content alginate and low molecular weight chitosan have been used as hydrogel carrier. Peptide recovery analyses, FTIR studies and molecular modelling highlighted chemical interactions between peptide and hydrogel polymers. Ac2-26 resulted entrapped into chitosan hydrogel matrix that prevented its release, whereas such interaction in alginate hydrogel slowed down peptide diffusion enabling its sustained release till 72 h. In vivo wound healing studies conducted on mice dorsal wounds indicate that after the 9th day of post wounding Ac2-26/alginate hydrogels could significantly accelerate wound healing, with complete closure of the wound on day 14th. Therefore, these results suggest that the developed of Ac2-26 high M content alginate hydrogel could be a promising wound dressing with potential application in dermal wound healing.

Asunto(s)

Anexina A1/administración & dosificación , Hidrogeles/administración & dosificación , Péptidos/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Administración Tópica , Alginatos/química , Animales , Anexina A1/química , Quitosano/química , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/química , Liberación de Fármacos , Hidrogeles/química , Inyecciones , Ratones Endogámicos C57BL , Péptidos/química

RESUMEN

BACKGROUND: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis. METHODS: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays. RESULTS: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells. CONCLUSION: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.

Asunto(s)

Anexina A1/metabolismo , Neoplasias Pancreáticas/patología , Anexina A1/farmacología , Línea Celular Tumoral , Movimiento Celular , Citoplasma/metabolismo , Transición Epitelial-Mesenquimal , Técnicas de Silenciamiento del Gen , Humanos , Invasividad Neoplásica , Neoplasias Pancreáticas/metabolismo , Péptidos/farmacología , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Neoplasias Pancreáticas

RESUMEN

Annexin A1 (ANXA1, lipocortin-1) is a glucocorticoid-regulated 37-kDa protein, so called since its main property is to bind (i.e. to annex) to cellular membranes in a Ca(2+)-dependent manner. Although ANXA1 has predominantly been studied in the context of immune responses and cancer, the protein can affect a larger variety of biological phenomena, including cell proliferation and migration. Our previous results show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. In this work, we have evaluated the hypothesis that ANXA1 is able to exert effects on myoblast cell migration acting through formyl peptide receptors (FPRs) following changes in its subcellular localization as in other cell types and tissues. The analysis of the subcellular localization of ANXA1 in C2C12 myoblasts during myogenic differentiation showed an interesting increase of extracellular ANXA1 starting from the initial phases of skeletal muscle cell differentiation. The investigation of intracellular Ca(2+) perturbation following exogenous administration of the ANXA1 N-terminal derived peptide Ac2-26 established the engagement of the FPRs which expression in C2C12 cells was assessed by qualitative PCR. Wound healing assay experiments showed that Ac2-26 peptide is able to increase migration of C2C12 skeletal muscle cells and to induce cell surface translocation and secretion of ANXA1. Our results suggest a role for ANXA1 as a highly versatile component in the signaling chains triggered by the proper calcium perturbation that takes place during active migration and differentiation or membrane repair since the protein is strongly redistributed onto the plasma membranes after an rapid increase of intracellular levels of Ca(2+). These properties indicate that ANXA1 may be involved in a novel repair mechanism for skeletal muscle and may have therapeutic implications with respect to the development of ANXA1 mimetics.

Asunto(s)

Anexina A1/metabolismo , Movimiento Celular/fisiología , Mioblastos/metabolismo , Receptores de Formil Péptido/metabolismo , Animales , Anexina A1/química , Anexina A1/farmacología , Western Blotting , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Movimiento Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Ratones , Microscopía Confocal , Proteína MioD/metabolismo , Mioblastos/citología , Miogenina/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Receptores de Formil Péptido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG) or high glucose (HG) we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i) Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii) N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.

Asunto(s)

Anexina A1/química , Movimiento Celular/efectos de los fármacos , Glucosa/administración & dosificación , Péptidos/farmacología , Anexina A1/farmacología , Señalización del Calcio , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Citometría de Flujo , Humanos

RESUMEN

Annexin A1 (ANXA1, lipocortin-1) is the first characterized member of the annexin superfamily of proteins, so called since their main property is to bind (i.e., to annex) to cellular membranes in a Ca(2+) -dependent manner. ANXA1 has been involved in a broad range of molecular and cellular processes, including anti-inflammatory signalling, kinase activities in signal transduction, maintenance of cytoskeleton and extracellular matrix integrity, tissue growth, apoptosis, and differentiation. New insights show that endogenous ANXA1 positively modulates myoblast cell differentiation by promoting migration of satellite cells and, consequently, skeletal muscle differentiation. This suggests that ANXA1 may contribute to the regeneration of skeletal muscle tissue and may have therapeutic implications with respect to the development of ANXA1 mimetics.

Asunto(s)

Anexina A1/metabolismo , Anexina A1/fisiología , Músculo Esquelético/crecimiento & desarrollo , Regeneración , Animales , Calcio/metabolismo , Diferenciación Celular , Movimiento Celular , Citoesqueleto/metabolismo , Humanos , Ratones , Músculo Esquelético/metabolismo , Transducción de Señal

RESUMEN

As a part of our drug discovery efforts we developed a series of simplified derivatives of bolinaquinone (BLQ), a hydroxyquinone marine metabolite, showing potent anti-inflammatory activity. Thirteen new hydroxyquinone derivatives closely related to BLQ were synthesized and tested on mouse macrophage-like RAW 264.7 cell line in order to investigate their ability to modulate the production of Prostaglandin E2 (PGE2). This optimization process led to the identification of three strictly correlated compounds with comparable and higher inhibitory potency than BLQ on PGE2 production. To evaluate the affinity of BLQ and its analogues for hsPLA2, surface plasmon resonance (SPR) experiments were performed.

Asunto(s)

Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacología , Diseño de Fármacos , Sesquiterpenos/química , Sesquiterpenos/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Línea Celular , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie

RESUMEN

Annexin A1 (ANXA1) is a calcium- and phospholipid-binding protein involved in a broad range of cellular events. This study used molecular and microscopy approaches to explore the role of ANXA1 in mouse myoblast C2C12 cell differentiation. We report that ANXA1 expression increases during differentiation and that the down-regulation of ANXA1 significantly inhibits the differentiation process. ANXA1 is expressed in vivo in both quiescent and activated satellite cells and is highly localized in the cells that migrate in the lumen of regenerating fibers after an acute injury. Endogenous ANXA1 co-localizes with actin fibers at the protruding ends of undifferentiated but not differentiated cells suggesting a role of the protein in cell migration. Furthermore, ANXA1 neutralizing antibody reduces MyHC expression, decreases myotube formation and significantly inhibits cell migration. The data reported here suggest for the first time that ANXA1 plays a role in myogenic differentiation.

Asunto(s)

Anexina A1/metabolismo , Diferenciación Celular/fisiología , Mioblastos/fisiología , Animales , Anexina A1/genética , Línea Celular , Ratones , Ratones Transgénicos , Desarrollo de Músculos/fisiología , Mioblastos/citología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/fisiología

RESUMEN

BACKGROUND: It has been shown that chemo-therapeutic agents, such as histone deacetylase inhibitors (HDACi), are able to increase TRAIL-induced apoptosis in many types of cancer. In the present study, we investigated the effects of the novel HDACi LGP1, a new simplified analogue of FR235222, in human leukaemia U937 cells resistant to TRAIL-induced apoptosis. MATERIALS AND METHODS: U937 cells were incubated with TRAIL/LGP1 for 24 h and apoptosis was evaluated using flow cytometric assay and cleavage of caspase-3 and (PARP) by Western blot. Western blot analysis was also used to detect the expression of p21, p27, (NF-kappaB), Bcl-2 and the levels of H4 histone acetylation. Finally, flow cytometry was used to monitor the enhancement of TRAIL-receptor levels. RESULTS: Treatment with LGP1 caused accumulation of acetylated histone H4 and G(1) cycle arrest accompanied by increase of p21. The compound was also able to sensitize U937 cells to TRAIL-induced apoptosis through multiple mechanisms: (i) activation of caspase-3 and cleavage of PARP; (ii) induction of p21 and p27; (iii) cleavage of NF-kappaB and down-regulation of Bcl-2. Finally, LGP1 induced up-regulation of TRAIL-R1 receptor expression. CONCLUSION: These results demonstrate that U937 cells can be effectively killed by a combination treatment of subtoxic doses of LGP1 and TRAIL.

Asunto(s)

Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Sinergismo Farmacológico , Activación Enzimática , Fase G1/efectos de los fármacos , Histonas/metabolismo , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Péptidos Cíclicos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Células U937