RESUMEN
The reduction of oxygen partial pressure in growing tumors triggers numerous survival strategies driven by the transcription factor complex HIF1 (Hypoxia Inducible Factor-1). Recent evidence revealed that HIF1 promotes rapid and effective phenotypic changes through the induction of non-coding RNAs, whose contribution has not yet been fully described. Here we investigated the role of the hypoxia-induced, long non-coding RNA H19 (lncH19) and its intragenic miRNA (miR-675-5p) into HIF1-Wnt crosstalk. During hypoxic stimulation, colorectal cancer cell lines up-regulated the levels of both the lncH19 and its intragenic miR-675-5p. Loss of expression experiments revealed that miR-675-5p inhibition, in hypoxic cells, hampered ß-catenin nuclear localization and its transcriptional activity, while lncH19 silencing did not induce the same effects. Interestingly, our data revealed that miRNA inhibition in hypoxic cells restored the activity of Glycogen Synthase Kinase 3ß (GSK-3ß) reducing the amount of P-Ser9 kinase, thus unveiling a role of the miR-675-5p in controlling GSK-3ß activity. Bioinformatics analyses highlighted the serine/threonine-protein phosphatases PPP2CA, responsible for GSK-3ß activation, among the miR-675-5p targets, thus indicating the molecular mediator through which miR-675-5p may control ß-catenin nuclear localization. In conclusion, here we demonstrated that the inhibition of the hypoxia-induced non-coding RNA miR-675-5p hampered the nuclear localization of ß-catenin by regulating GSK-3ß activity, thus proposing the miR-675-5p as a new therapeutic target for the treatment of colorectal cancer.
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Hipoxia de la Célula , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , MicroARNs/metabolismo , beta Catenina/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Biología Computacional , Células HCT116 , Humanos , Estimación de Kaplan-Meier , Microscopía Fluorescente , Mutación , Unión Proteica , TransfecciónRESUMEN
In recent years, several studies demonstrated the role of exosomes in intercellular communications, several Leishmania species belonging to subgenera Leishmania and Viannia have been demonstrated to release exosomes, and their role in parasite-macrophage interactions and in leishmaniasis development has been investigated. However, the release of exosomes by Leishmania infantum has not been studied so far. The aim of this study was to isolate and characterize L. infantum exosomes, and to investigate the biological activity of these exosomes in macrophage cultures. To this end, exosomes were collected from both amastigote and promastigote L. infantum conditioned medium by ultracentrifugation. Exosomes were then characterized by monitoring the presence of HSP70, HSP83/90 and acetylcholinesterase activity. Moreover, extracellular vesicles-tracking analysis revealed that promastigote and amastigote exosomes had mean diameter of 122⯱â¯56â¯nm and 115⯱â¯65â¯nm, respectively. Human monocytic cell line U937-derived macrophages treated with promastigote and amastigote exosomes showed an increase in motility and an overproduction of interleukin IL-10 and IL-18 reduction, involved in immune response. Since L. infantum exosomes demonstrated the capacity to modulate the chemotactic behaviour of the cells studied and cytokines production, they could contribute in the disease establishment and may be considered an appropriate candidate for a vaccine therapy in prophylaxis and treatment.
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Quimiotaxis/fisiología , Citocinas/metabolismo , Exosomas/metabolismo , Leishmania infantum/metabolismo , Citocinas/genética , Expresión Génica , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-18/metabolismo , Células U937RESUMEN
BACKGROUND: Multiple myeloma (MM) is a clonal plasma cell malignancy associated with osteolytic bone disease. Recently, the role of MM-derived exosomes in the osteoclastogenesis has been demonstrated although the underlying mechanism is still unknown. Since exosomes-derived epidermal growth factor receptor ligands (EGFR) are involved in tumor-associated osteolysis, we hypothesize that the EGFR ligand amphiregulin (AREG) can be delivered by MM-derived exosomes and participate in MM-induced osteoclastogenesis. METHODS: Exosomes were isolated from the conditioned medium of MM1.S cell line and from bone marrow (BM) plasma samples of MM patients. The murine cell line RAW264.7 and primary human CD14+ cells were used as osteoclast (OC) sources. RESULTS: We found that AREG was specifically enriched in exosomes from MM samples and that exosomes-derived AREG led to the activation of EGFR in pre-OC, as showed by the increase of mRNA expression of its downstream SNAIL in both RAW264.7 and CD14+ cells. The presence of neutralizing anti-AREG monoclonal antibody (mAb) reverted this effect. Consequently, we showed that the effect of MM-derived exosomes on osteoclast differentiation was inhibited by the pre-treatment of exosomes with anti-AREG mAb. In addition, we demonstrated the ability of MM-derived AREG-enriched exosomes to be internalized into human mesenchymal stromal cells (MSCs) blocking osteoblast (OB) differentiation, increasing MM cell adhesion and the release of the pro-osteoclastogenic cytokine interleukin-8 (IL8). Accordingly, anti-AREG mAb inhibited the release of IL8 by MSCs suggesting that both direct and indirect effects are responsible for AREG-enriched exosomes involvement on MM-induced osteoclastogenesis. CONCLUSIONS: In conclusion, our data indicate that AREG is packed into MM-derived exosomes and implicated in OC differentiation through an indirect mechanism mediated by OBs.
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Anfirregulina/genética , Factor de Crecimiento Epidérmico/metabolismo , Exosomas/metabolismo , Mieloma Múltiple/metabolismo , Osteogénesis , Microambiente Tumoral , Anfirregulina/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Adhesión Celular , Diferenciación Celular , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Interleucina-8/antagonistas & inhibidores , Ligandos , Células Madre Mesenquimatosas , Ratones , Mieloma Múltiple/patología , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Células RAW 264.7RESUMEN
BACKGROUND: We aimed to evaluate the phenotype, function, and microRNA (miRNA)17-92 cluster expression in Vγ9Vδ2 T-cell subsets and the correlation with immune response in rheumatoid arthritis (RA) patients. METHODS: Peripheral blood from 10 early RA untreated patients and 10 healthy donors (HD) was obtained. Polyclonal Vγ9Vδ2 T-cell lines were generated and analysed by flow cytometry. Analysis of miRNA17-92 cluster expression was performed by real-time polymerase chain reaction (RT-PCR), and expression of mRNA target genes was also studied. RESULTS: A remarkable change in the distribution of Vγ9Vδ2 T-cell functional subsets was observed in the peripheral blood of RA patients compared with HD, with an expansion of effector subsets and reduction of naive cells which was accompanied by modifications in proinflammatory cytokine expression. Vγ9Vδ2 T cells with a TEM (effector memory) phenotype and producing proinflammatory cytokines were correlated with disease activity score (DAS28). The comparison of miRNA expression among Vγ9Vδ2 T-cell subsets from RA patients and HD showed a lower level of miR-106a-5p and miR-20a-5p, and a higher level of miR-21a-5p, among Vγ9Vδ2 TEM cells, and a lower level of miR-19b-3p among Vγ9Vδ2 TCM (central memory) cells was also found. These differentially expressed miRNAs correlated with higher levels of expression of interleukin (IL)-8, IL-6, and PDCD4 genes. CONCLUSIONS: Our results provide evidence for a role of miR-106a, miR-19-3p, miR-20a, and miR-21a in the regulation of Vγ9Vδ2 T-cell function in RA patients and suggest the possibility that the miRNA17-92 family and Vγ9Vδ2 T cells contribute to the pathogenesis of RA.
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Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Regulación hacia Abajo/fisiología , MicroARNs/biosíntesis , Subgrupos de Linfocitos T/metabolismo , Adulto , Artritis Reumatoide/genética , Femenino , Humanos , Masculino , MicroARNs/genética , Persona de Mediana EdadRESUMEN
OBJECTIVE: Gut-derived innate lymphoid cell 3 (ILC3) has been shown to participate in the pathogenesis of ankylosing spondylitis (AS). CX3 CR1+ mononuclear phagocytes (MNPs) have been demonstrated to modulate ILC3 function in the gut. This study was undertaken to investigate the role of proinflammatory CX3 CR1+CD59+ MNPs in modulating ILC3 function in AS patients. METHODS: MNP subsets in the blood of AS patients and controls were analyzed by flow cytometry. The presence of CX3 CR1+CD59+ cells in tissue was confirmed by confocal microscopy. Expression of the proinflammatory chemokines CX3 CL1 and CCL2 and decoy receptor 6 (DcR-6) was analyzed. Peripheral CX3 CR1+CD59+ cells were cocultured with ILC3, and changes in their frequency were evaluated by flow cytometry. Transcriptome analysis of circulating CX3 CR1+ monocytes was also performed. RESULTS: DcR-6 deficiency and CCL2 overexpression were observed in inflamed tissues from AS patients. In the gut, the proinflammatory CX3 CR1+CD59+ MNP population was expanded, correlated with the presence of bacteria, and produced high levels of tumor necrosis factor-like molecule 1A (TL1A) and interleukin-23 (IL-23). MNPs positive for CD11b, CD11c, and major histocompatibility complex class II, predominantly expressing CX3 CR1, were also expanded in the small intestines of treatment-naive SKG relative to BALB/c mice. The frequency of gut-derived CX3 CR1+CD59+CCR9+TL1A+IL-23+ MNPs was significantly higher in the peripheral blood and synovial fluid of AS patients than controls. CCR9+CX3 CR1+CD59+ monocytes were also expanded in AS synovial and bone marrow samples. Transcriptome analysis of isolated CX3 CR1+CD59+ monocytes demonstrated a specific proinflammatory profile in AS. Isolated proinflammatory CX3 CR1+CD59+ MNPs from AS patients induced the expansion and activation of ILC3. CONCLUSION: Proinflammatory CX3 CR1+CD59+TL1A+IL-23+ MNPs are expanded in AS patients and display a specific proinflammatory transcriptome profile. Given the ability of these cells to support ILC3 expansion, they may promote a sustained proinflammatory status in AS.
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Inmunidad Innata , Linfocitos/inmunología , Monocitos/inmunología , Sistema Mononuclear Fagocítico/inmunología , Espondilitis Anquilosante/inmunología , Adulto , Antígenos CD59/inmunología , Receptor 1 de Quimiocinas CX3C/inmunología , Estudios de Casos y Controles , Femenino , Humanos , Interleucina-23/inmunología , Masculino , Persona de Mediana Edad , Receptores Tipo I de Factores de Necrosis Tumoral/inmunologíaRESUMEN
BACKGROUND: Our previous study demonstrates that Citrus-limon derived nanovesicles are able to decrease colon cancer cell viability, and that this effect is associated with the downregulation of the intracellular phospholipase DDHD domain-containing protein 1 (DDHD1). While few studies are currently available on the contribution of DDHD1 in neurological disorders, there is no information on its role in cancer. This study investigates the role of DDHD1 in colon cancer. METHODS: DDHD1 siRNAs and an overexpression vector were transfected into colorectal cancer and normal cells to downregulate or upregulate DDHD1 expression. In vitro and in vivo assays were performed to investigate the functional role of DDHD1 in colorectal cancer cell growth. Quantitative proteomics using SWATH-MS was performed to determinate the molecular effects induced by DDHD1 silencing in colorectal cancer cells. RESULTS: The results indicate that DDHD1 supports colon cancer cell proliferation and survival, since its downregulation reduces in vitro colon cancer cell viability and increases apoptosis rate, without affecting normal cells. On the contrary, in vivo studies demonstrate that the xenograft tumors, derived from DDHD1-overexpressing cells, have a higher proliferation rate compared to control animals. Additionally, we found that functional categories, significantly affected by DDHD1 silencing, were specifically related to cancer phenotype and for the first time associated to DDHD1 activity. CONCLUSIONS: In conclusion, this study provides the first evidence confirming the role of DDHD1 in cancer, providing a possibility to define a new target to design more effective therapies for colon cancer patients.
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Antineoplásicos/farmacología , Biomarcadores de Tumor , Neoplasias Colorrectales/metabolismo , Terapia Molecular Dirigida , Fosfolipasas/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Biología Computacional/métodos , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Silenciador del Gen , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Fosfolipasas/genética , Fosfolipasas/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To investigate the role of the interleukin-25 (IL-25)/IL-17 receptor B (IL-17RB) axis in experimental Sjögren's syndrome (SS) and in patients with primary SS and primary SS-associated lymphoma. METHODS: Expression of IL-25, IL-17RB, IL-17B, and tumor necrosis factor receptor-associated factor 6 (TRAF6) was analyzed on minor salivary gland (SG) samples from patients with primary SS and on parotid gland samples from patients with primary SS-associated B cell non-Hodgkin's lymphoma (NHL). IL-17RB expression and the frequencies of natural group 2 innate lymphoid cells (ILC2s), inflammatory ILC2s, and M2-polarized macrophages were assessed by flow cytometry in SG mononuclear cells and peripheral blood mononuclear cells (PBMCs). Tissue distribution of ILC2s was studied by confocal microscopy. The role of recombinant IL-25 and of rituximab in modulating IL-25 expression was investigated in in vitro studies. IL-25/IL-17RB and TRAF6 expression and the role of IL-25 inhibition were also studied in the experimental murine model of SS. RESULTS: Activation of the IL-25/IL-17RB/TRAF6 axis correlated with the focus score and was observed in patients with primary SS and in patients with primary SS-associated NHL. A significant increase in the frequency of inflammatory ILC2s was observed both in SG mononuclear cells and in PBMCs. IL-25 stimulation of isolated SG mononuclear cells and PBMCs from patients and controls resulted both in inflammatory ILC2 expansion and in increased autoantibody production. Rituximab modulated expression of inflammatory ILC2s and IL-25 in primary SS. SG protein-immunized mice developed overt SS symptoms with increased IL-25 expression and increased frequency of CD4+IL-17RB+TRAF6+ cells. IL-25 neutralization attenuated disease progression and tissue pathology in mice with experimental SS. CONCLUSION: IL-25 may promote the inflammatory state in primary SS and may be a potential target for novel disease-modifying therapeutic strategies in patients with primary SS.
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Interleucina-17/metabolismo , Linfoma/inmunología , Receptores de Interleucina-17/metabolismo , Síndrome de Sjögren/inmunología , Anciano , Animales , Femenino , Humanos , Inmunidad Innata , Leucocitos Mononucleares/inmunología , Linfocitos/inmunología , Macrófagos/inmunología , Masculino , Ratones , Persona de Mediana Edad , Glándulas Salivales/inmunologíaRESUMEN
We have previously isolated exosome-like nanoparticles from Citrus-limon juice, able to inhibit in vitro and in vivo tumor cell growth. In order to deeply understand the mechanism underlying nanovesicle effects, we performed a proteomic profile of treated colorectal cancer cells. Among the proteins differentially expressed after nanovesicle treatment, we found a significant downregulation of the Acetyl-CoA Carboxylase 1 (ACACA) and we demonstrated that silencing ACACA in cancer cells leads to a reduction of cell growth. Our study proved that the anti-tumor effects of Citrus-limon nanovesicles is partly mediated by lipid metabolism inhibition, in particular via ACACA downregulation. SIGNIFICANCE: This study represents the attempt to achieve, by a proteomic approach, a better understanding of the role of lemon nanovesicles in affecting colorectal cancer cell growth.
Asunto(s)
Acetil-CoA Carboxilasa/antagonistas & inhibidores , Citrus/toxicidad , Neoplasias del Colon/tratamiento farmacológico , Exosomas/química , Proteómica/métodos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Exosomas/fisiología , Humanos , Metabolismo de los Lípidos/efectos de los fármacosRESUMEN
MAIT cells are expanded in salivary glands of patients with Sjogren's syndrome and are IL-17 polarized. IL-7 and IL-23 induce IL-17 production activating two different pathways: IL-7 stimulation induces in fact a significant STAT3 and HIF1alpha upregulation, conversely, IL-23 stimulation significantly induces RORc overexpression in MAIT cells of patients with Sjogren's syndrome.
Asunto(s)
Interleucina-17 , Células T Invariantes Asociadas a Mucosa , Humanos , Interleucina-23 , Glándulas Salivales , Síndrome de SjögrenRESUMEN
A fundamental task in cancer research aims at the identification of new pharmacological therapies that can affect tumor growth. Differentiation therapy might exploit this function not only for hematological diseases, such as acute promyelocytic leukemia (APML) but also for epithelial tumors, including lung cancer. Here we show that Retinoic Acid (RA) arrests in vitro and in vivo the growth of Tyrosine Kinase Inhibitors (TKI) resistant Non Small Cell Lung Cancer (NSCLC). In particular, we found that RA induces G0/G1 cell cycle arrest in TKI resistant NSCLC cells and activates terminal differentiation programs by modulating the expression of GATA6, a key transcription factor involved in the physiological differentiation of the distal lung. In addition, our results demonstrate that RA inhibits EGFR and Wnt signaling activation, two pathways involved in NSCLC progression. Furthermore, we uncovered a novel mechanism in NSCLC that shows how RA exerts its function; we found that RA-mediated GATA6 activation is necessary for EGFR and Wnt inhibition, thus leading to 1) increased differentiation and 2) loss of proliferation. All together, these findings prove that differentiation therapy might be feasible in TKI resistant NSCLCs, and shed light on new targets to define new pharmacological therapies.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/efectos de los fármacos , Factor de Transcripción GATA6/efectos de los fármacos , Tretinoina/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Factor de Transcripción GATA6/metabolismo , Humanos , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. METHODS: Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. RESULTS: Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. CONCLUSIONS: Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour.
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Toxina del Cólera/sangre , Disbiosis/inmunología , Endotelio/metabolismo , Ileítis/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Espondilitis Anquilosante/inmunología , Enfermedad Aguda , Proteínas de Fase Aguda , Uniones Adherentes/genética , Animales , Antibacterianos/farmacología , Antígenos CD/genética , Bacterias/aislamiento & purificación , Células CACO-2 , Cadherinas/genética , Proteínas Portadoras/sangre , Proteínas Portadoras/genética , Estudios de Casos y Controles , Toxina del Cólera/genética , Enfermedad Crónica , Disbiosis/microbiología , Proteínas de Unión a Ácidos Grasos/sangre , Expresión Génica , Antígeno HLA-B27/genética , Haptoglobinas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ileítis/sangre , Íleon/inmunología , Íleon/microbiología , Interleucina-8 , Mucosa Intestinal/microbiología , Molécula A de Adhesión de Unión/genética , Lipopolisacáridos/sangre , Glicoproteínas de Membrana/sangre , Proteínas de la Membrana/genética , Monocitos/inmunología , Permeabilidad , Precursores de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Transgénicas , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/genética , Regulación hacia ArribaRESUMEN
Chronic myelogenous leukaemia (CML) is a clonal myeloproliferative disorder. Recent evidence indicates that altered crosstalk between CML and mesenchymal stromal cells may affect leukaemia survival; moreover, vesicles released by both tumour and non-tumour cells into the microenvironment provide a suitable niche for cancer cell growth and survival. We previously demonstrated that leukaemic and stromal cells establish an exosome-mediated bidirectional crosstalk leading to the production of IL8 in stromal cells, thus sustaining the survival of CML cells. Human cell lines used are LAMA84 (CML cells), HS5 (stromal cells) and bone marrow primary stromal cells; gene expression and protein analysis were performed by real-time PCR and Western blot. IL8 and MMP9 secretions were evaluated by ELISA. Exosomes were isolated from CML cells and blood samples of CML patients. Here, we show that LAMA84 and CML patients' exosomes contain amphiregulin (AREG), thus activating epidermal growth factor receptor (EGFR) signalling in stromal cells. EGFR signalling increases the expression of SNAIL and its targets, MMP9 and IL8. We also demonstrated that pre-treatment of HS5 with LAMA84 exosomes increases the expression of annexin A2 that promotes the adhesion of leukaemic cells to the stromal monolayer, finally supporting the growth and invasiveness of leukaemic cells. Leukaemic and stromal cells establish a bidirectional crosstalk: exosomes promote proliferation and survival of leukaemic cells, both in vitro and in vivo, by inducing IL8 secretion from stromal cells. We propose that this mechanism is activated by a ligand-receptor interaction between AREG, found in CML exosomes, and EGFR in bone marrow stromal cells.
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Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Microambiente Celular , Receptores ErbB/metabolismo , Exosomas/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Anfirregulina/metabolismo , Anexina A2/metabolismo , Adhesión Celular , Línea Celular Tumoral , Humanos , Interleucina-8/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismo , Células del Estroma/metabolismoRESUMEN
Tumor derived exosomes are vesicles which contain proteins and microRNAs that mediate cell-cell communication and are involved in angiogenesis and tumor progression. Curcumin derived from the plant Curcuma longa, shows anticancer effects. Exosomes released by CML cells treated with Curcumin contain a high amount of miR-21 that is shuttled into the endothelial cells in a biologically active form. The treatment of HUVECs with CML Curcu-exosomes reduced RhoB expression and negatively modulated endothelial cells motility. We showed that the addition of CML control exosomes to HUVECs caused an increase in IL8 and VCAM1 levels, but Curcu-exosomes reversed these effects thus attenuating their angiogenic properties. This antiangiogenic effect was confirmed with in vitro and in vivo vascular network formation assays. SWATH analysis of the proteomic profile of Curcu-exosomes revealed that Curcumin treatment deeply changes their molecular properties, in particular, Curcumin induces a release of exosomes depleted in pro-angiogenic proteins and enriched in proteins endowed with anti-angiogenic activity. Among the proteins differential expressed we focused on MARCKS, since it was the most modulated protein and a target of miR-21. Taken together our data indicated that also Curcumin attenuates the exosome's ability to promote the angiogenic phenotype and to modulate the endothelial barrier organization.
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Curcumina/farmacología , Exosomas/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , MicroARNs/genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Células Cultivadas , Exosomas/genética , Exosomas/metabolismo , Regulación de la Expresión Génica , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodosRESUMEN
BACKGROUND: CD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes. METHODS: Exosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments. RESULTS: Exosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells. CONCLUSIONS: Our data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.
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Células Endoteliales/metabolismo , Exosomas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Fenotipo , ARN Largo no Codificante/genética , Antígenos Thy-1/metabolismo , Adhesión Celular , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana/metabolismo , HumanosRESUMEN
Nanosized vesicles are considered key players in cell to cell communication, thus influencing physiological and pathological processes, including cancer. Nanovesicles have also been found in edible-plants and have shown therapeutic activity in inflammatory bowel diseases; however information on their role in affecting cancer progression is missing.Our study identify for the first time a fraction of vesicles from lemon juice (Citrus limon L.), obtained as a result of different ultracentrifugation, with density ranging from 1,15 to 1,19 g/ml and specific proteomic profile. By using an in vitro approach, we show that isolated nanovesicles inhibit cancer cell proliferation in different tumor cell lines, by activating a TRAIL-mediated apoptotic cell death. Furthermore, we demonstrate that lemon nanovesicles suppress CML tumor growth in vivo by specifically reaching tumor site and by activating TRAIL-mediated apoptotic cell processes. Overall, this study suggests the possible use of plant-edible nanovesicles as a feasible approach in cancer treatment.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citrus , Exosomas , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Nanopartículas , Extractos Vegetales/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Carga Tumoral/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citrus/química , Exosomas/química , Exosomas/metabolismo , Jugos de Frutas y Vegetales , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones Endogámicos NOD , Ratones SCID , Fitoterapia , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/metabolismo , Proteínas de Plantas/análisis , Plantas Medicinales , Proteómica/métodos , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS). METHODS: ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed. RESULTS: ILC3 characterised as Lyn(-)RORc(-)Tbet(+) NKp44(+) cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed α4ß7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R(+) cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1. CONCLUSIONS: Gut-derived IL-17(+) and IL-22(+)ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints.
Asunto(s)
Médula Ósea/inmunología , Mucosa Intestinal/inmunología , Linfocitos/inmunología , Espondilitis Anquilosante/inmunología , Líquido Sinovial/inmunología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Moléculas de Adhesión Celular , Femenino , Humanos , Íleon/inmunología , Íleon/patología , Inmunoglobulinas/metabolismo , Interleucina-15/inmunología , Interleucina-17/inmunología , Interleucina-7/inmunología , Interleucinas/inmunología , Masculino , Microscopía Confocal , Persona de Mediana Edad , Mucoproteínas/metabolismo , Receptor 2 Gatillante de la Citotoxidad Natural , Adulto Joven , Interleucina-22RESUMEN
BACKGROUND: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder in which leukemic cells display a reciprocal t(9:22) chromosomal translocation that results in the formation of the chimeric BCR-ABL oncoprotein, with a constitutive tyrosine kinase activity. Consequently, BCR-ABL causes increased proliferation, inhibition of apoptosis, and altered adhesion of leukemic blasts to the bone marrow (BM) microenvironment. It has been well documented that cancer cells can generate their own signals in order to sustain their growth and survival, and recent studies have revealed the role of cancer-derived exosomes in activating signal transduction pathways involved in cancer cell proliferation. Exosomes are small vesicles of 40-100 nm in diameter that are initially formed within the endosomal compartment, and are secreted when a multivesicular body (MVB) fuses with the plasma membrane. These vesicles are released by many cell types including cancer cells, and are considered messengers in intercellular communication. We have previously shown that CML cells released exosomes able to affect the tumor microenvironment. RESULTS: CML cells, exposed up to one week, to exosomes showed a dose-dependent increased proliferation compared with controls. Moreover, exosome treatment promotes the formation of LAMA84 colonies in methylcellulose. In a CML xenograft model, treatment of mice with exosomes caused a greater increase in tumor size compared with controls (PBS-treated mice). Real time PCR and Western Blot analysis showed, in both in vitro and in vivo samples, an increase in mRNA and protein levels of anti-apoptotic molecules, such as BCL-w, BCL-xl, and survivin, and a reduction of the pro-apoptotic molecules BAD, BAX and PUMA. We also found that TGF- ß1 was enriched in CML-exosomes. Our investigations showed that exosome-stimulated proliferation of leukemia cells, as well as the exosome-mediated activation of an anti-apoptotic phenotype, can be inhibited by blocking TGF-ß1 signaling. CONCLUSIONS: CML-derived exosomes promote, through an autocrine mechanism, the proliferation and survival of tumor cells, both in vitro and in vivo, by activating anti-apoptotic pathways. We propose that this mechanism is activated by a ligand-receptor interaction between TGF-ß1, found in CML-derived exosomes, and the TGF- ß1 receptor in CML cells.
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Comunicación Autocrina , Proliferación Celular , Exosomas/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Transducción de Señal , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Exosomas/genética , Exosomas/patología , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones Desnudos , Ratones SCID , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Survivin , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: Recent findings indicate that exosomes released from cancer cells contain microRNAs (miRNAs) that may be delivered to cells of tumor microenvironment. RESULTS: To elucidate whether miRNAs secreted from chronic myelogenous leukemia cells (CML) are shuttled into endothelial cells thus affecting their phenotype, we first analysed miRNAs content in LAMA84 exosomes. Among the 124 miRNAs identified in LAMA84 exosomes, we focused our attention on miR-126 which was found to be over-overexpressed in exosomes compared with producing parental cells. Transfection of LAMA84 with Cy3-labelled miR-126 and co-culture of leukemia cells with endothelial cells (EC) confirmed that miR-126 is shuttled into HUVECs. The treatment of HUVECs with LAMA84 exosomes for 24 hours reduced CXCL12 and VCAM1 expression, both at the mRNA and protein level, and negatively modulated LAMA84 motility and cells adhesion. Transfection in HUVECs of miR-126 inhibitor reversed the decrease of CXCL12 and restored the motility and adhesion of LAMA84 cells while the over-expression of miR-126, showed opposite effects. CONCLUSION: Our results show that the miR-126 shuttled by exosomes is biologically active in the target cells, and support the hypothesis that exosomal miRNAs have an important role in tumor-endothelial crosstalk occurring in the bone marrow microenvironment, potentially affecting disease progression.
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Exosomas/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , MicroARNs/genética , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Quimiocina CXCL12/biosíntesis , Células Endoteliales/metabolismo , Exosomas/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the Bcr-Abl oncoprotein with constitutive tyrosine kinase activity. Exosomes are nanovesicles released by cancer cells that are involved in cell-to-cell communication thus potentially affecting cancer progression. It is well known that bone marrow stromal microenvironment contributes to disease progression through the establishment of a bi-directional crosstalk with cancer cells. Our hypothesis is that exosomes could have a functional role in this crosstalk. Interleukin-8 (IL 8) is a proinflammatory chemokine that activates multiple signalling pathways downstream of two receptors (CXCR1 and CXCR2). We demonstrated that exosomes released from CML cells stimulate bone marrow stromal cells to produce IL 8 that, in turn, is able to modulate both in vitro and in vivo the leukemia cell malignant phenotype.
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Exosomas/metabolismo , Interleucina-8/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Células Madre Mesenquimatosas/metabolismo , Comunicación Paracrina , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Xenoinjertos , Humanos , Interleucina-8/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fenotipo , Transducción de Señal , Nicho de Células Madre , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
OBJECTIVE: The aim of this study was to assess the expression of IL-22, IL-22 receptor 1 (IL-22R1), IL-22 binding protein (IL-22BP) and p-STAT3 in muscle tissue from patients with PM and DM. METHODS: Levels of IL-22, IL-22R1, IL-22BP and STAT3 mRNA were quantified by RT-PCR. The expression of IL-22, IL-22R1, IL-22BP and p-STAT3 was also analysed using immunohistochemistry. RESULTS: Significant modulation of the IL-22 pathway was observed in inflammatory myopathic tissues. In particular, a significant overexpression of IL-22 at the protein but not the mRNA level was observed in PM/DM tissues and was correlated with myositis activity. IL-22R1 aberrant expression was also observed among infiltrating mononuclear cells and necrotic muscle cells. IL-22BP, which inhibits IL-22 signalling, was expressed only in some muscle fibres in PM/DM patients. CONCLUSION: Our findings indicate that the IL-22 pathway is activated in inflammatory myopathic tissues and may be involved in the induction of muscle inflammatory processes and muscle necrosis.