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Despite the tremendous advances that have been made in biomedical research, cancer remains one of the leading causes of death worldwide. Several therapeutic approaches have been suggested and applied to treat cancer with impressive results. Immunotherapy based on targeting immune checkpoint signaling pathways proved to be one of the most efficient. In this review article, we will focus on the recently discovered TNFα-TNFR2 signaling pathway, which controls the immunological and pro-angiogenic properties of many immunoregulatory and pro-angiogenic cells such as endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and regulatory T cells (Tregs). Due to their biological properties, these cells can play a major role in cancer progression and metastasis. Therefore, we will discuss the advantages and disadvantages of an anti-TNFR2 treatment that could carry two faces under one hood. It interrupts the immunosuppressive and pro-angiogenic behaviors of the above-mentioned cells and interferes with tumor growth and survival.
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BACKGROUND: Moyamoya angiopathy (MMA) is a rare cerebrovascular condition leading to stroke. Mutations in 15 genes have been identified in Mendelian forms of MMA, but they explain only a very small proportion of cases. Our aim was to investigate the genetic basis of MMA in consanguineous patients having unaffected parents in order to identify genes involved in autosomal recessive MMA. METHODS: Exome sequencing (ES) was performed in 6 consecutive consanguineous probands having MMA of unknown etiology. Functional consequences of variants were assessed using western blot and protein 3D structure analyses. RESULTS: Causative homozygous variants of NOS3, the gene encoding the endothelial nitric oxide synthase (eNOS), and GUCY1A3, the gene encoding the alpha1 subunit of the soluble guanylate cyclase (sGC) which is the major nitric oxide (NO) receptor in the vascular wall, were identified in 3 of the 6 probands. One NOS3 variant (c.1502 + 1G > C) involves a splice donor site causing a premature termination codon and leads to a total lack of eNOS in endothelial progenitor cells of the affected proband. The other NOS3 variant (c.1942 T > C) is a missense variant located into the flavodoxine reductase domain; it is predicted to be destabilizing and shown to be associated with a reduction of eNOS expression. The GUCY1A3 missense variant (c.1778G > A), located in the catalytic domain of the sGC, is predicted to disrupt the tridimensional structure of this domain and to lead to a loss of function of the enzyme. Both NOS3 mutated probands suffered from an infant-onset and severe MMA associated with posterior cerebral artery steno-occlusive lesions. The GUCY1A3 mutated proband presented an adult-onset MMA associated with an early-onset arterial hypertension and a stenosis of the superior mesenteric artery. None of the 3 probands had achalasia. CONCLUSIONS: We show for the first time that biallelic loss of function variants in NOS3 is responsible for MMA and that mutations in NOS3 and GUCY1A3 are causing fifty per cent of MMA in consanguineous patients. These data pinpoint the essential role of the NO pathway in MMA pathophysiology.
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Enfermedad de Moyamoya , Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico , Guanilil Ciclasa Soluble , Adulto , Humanos , Enfermedad de Moyamoya/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Transducción de Señal/genética , Guanilil Ciclasa Soluble/genéticaRESUMEN
Circulating endothelial progenitor cells (EPCs) play a pivotal role in the repair of diseases in which angiogenesis is required. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and, especially, long-term immune rejection. EPC-derived extracellular vesicles (EPC-EVs) may be an alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. Here, we investigated the regenerative effects of umbilical cord blood (CB) EPC-EVs on CB-EPCs in vitro. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then, EVs were isolated from the conditioned medium with tangential flow filtration (TFF). The regenerative effects of EVs on cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed their effects on endothelial cell inflammation and Nitric Oxide (NO) production. We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher dose than the physiological dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features. Our results reveal for the first time that EPC-EVs, when used at a high dose, enhance EPC regenerative functions without altering their endothelial identity.
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Células Progenitoras Endoteliales , Vesículas Extracelulares , Humanos , Células Progenitoras Endoteliales/metabolismo , Sangre Fetal , Inflamación/metabolismo , Movimiento Celular , Células CultivadasRESUMEN
The most widely used genome editing toolkit is CRISPR (clustered regularly interspaced short palindromic repeats). It provides the possibility of replacing and modifying DNA and RNA nucleotides. Furthermore, with advancements in biological technology, inhibition and activation of the transcription of specific gene(s) has become possible. Bioinformatics tools that target the evolution of CRISPR-associated protein 9 (Cas9) turn this protein into a vehicle that is specific for a DNA or RNA region with single guide RNA (sgRNA). This toolkit could be used by researchers to investigate the function of stem cell gene(s). Here, in this review article, we cover recent developments and applications of this technique in stem cells for research and clinical purposes and discuss different CRISPR/Cas technologies for knock-out, knock-in, activation, or inhibition of gene expression. Additionally, a comparison of several deliveries and off-target detecting strategies is discussed.
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Sistemas CRISPR-Cas , Edición Génica , Edición Génica/métodos , Células Madre , ARN , ADN/genéticaRESUMEN
Vero cells are one of the most frequently used cell types in virology. They can be used not only as a vehicle for the replication of viruses, but also as a model for investigating viral infectivity, cytopathology and vaccine production. There is increasing awareness of the need to limit the use of animal-derived components in cell culture media for a number of reasons, which include reducing the risk of contamination and decreasing costs related to the downstream processing of commercial products obtained via cell culture. The current study evaluates the use of protein hydrolysates (PHLs), also known as peptones, as partial substitutes for fetal bovine serum (FBS) in Vero cell culture. Eleven plant-based, two yeast-based, and three casein-based peptones were assessed, with different batches evaluated in the study. We tested the effects of three concentration ratios of FBS and peptone on Vero cell proliferation, four days after the initial cell seeding. Some of the tested peptones, when in combination with a minimal 1% level of FBS, supported cell proliferation rates equivalent to those achieved with 10% FBS. Collectively, our findings showed that plant-based peptones could represent promising options for the successful formulation of serum-reduced cell culture media for vaccine production. This is especially relevant in the context of the current COVID-19 pandemic, in view of the urgent need for SARS-CoV-2 virus production for certain types of vaccine. The current study contributes to the Three Rs principle of reduction, as well as addressing animal ethics concerns associated with FBS, by repurposing PHLs for use in cell culture.
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COVID-19 , Peptonas , Animales , Caseínas , Técnicas de Cultivo de Célula , Chlorocebus aethiops , Medios de Cultivo/farmacología , Humanos , Pandemias , Peptonas/metabolismo , Peptonas/farmacología , Hidrolisados de Proteína , SARS-CoV-2 , Albúmina Sérica Bovina , Células VeroRESUMEN
BACKGROUND: Bone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions. METHODS: EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect. RESULTS: Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions. CONCLUSIONS: We report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs' longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration. Video Abstract.
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Células Progenitoras Endoteliales/efectos de los fármacos , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Células Progenitoras Endoteliales/inmunología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Inmunomodulación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Skin grafting is a surgical method of cutaneous reconstruction, which provides volumetric replacement in wounds unable to heal by primary intention. Clinically, full-thickness skin grafts (FTSGs) are placed in aesthetically sensitive and mechanically demanding areas such as the hands, face, and neck. Complete or partial graft failure is the primary complication associated with this surgical procedure. Strategies aimed at improving the rate of skin graft integration will reduce the incidence of graft failure. Cold atmospheric plasma (CAP) is an emerging technology offering innovative clinical applications. The aim of this study was to test the therapeutic potential of CAP to improve wound healing and skin graft integration into the recipient site. In vitro models that mimic wound healing were used to investigate the ability of CAP to enhance cellular migration, a key factor in cutaneous tissue repair. We demonstrated that CAP enhanced the migration of epidermal keratinocytes and dermal fibroblasts. This increased cellular migration was possibly induced by the low dose of reactive oxygen and nitrogen species produced by CAP. Using a mouse model of burn wound reconstructed with a full-thickness skin graft, we showed that wounds treated with CAP healed faster than did control wounds. Immunohistochemical wound analysis showed that CAP treatment enhanced the expression of the dermal-epidermal junction components, which are vital for successful skin graft integration. CAP treatment was characterised by increased levels of Tgfbr1 mRNA and collagen I protein in vivo, suggesting enhanced wound maturity and extracellular matrix deposition. Mechanistically, we show that CAP induced the activation of the canonical SMAD-dependent TGF-ß1 pathway in primary human dermal fibroblasts, which may explain the increased collagen I synthesis in vitro. These studies revealed that CAP improved wound repair and skin graft integration via mechanisms involving extracellular matrix formation. CAP offers a novel approach for treating cutaneous wounds and skin grafts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Quemaduras/cirugía , Matriz Extracelular/fisiología , Queratinocitos/fisiología , Gases em Plasma/uso terapéutico , Repitelización/fisiología , Trasplante de Piel/métodos , Cicatrización de Heridas/fisiología , Animales , Quemaduras/fisiopatología , Movimiento Celular/fisiología , Proliferación Celular , Ratones , Modelos Animales , Fenómenos Fisiológicos de la Piel , Resultado del TratamientoRESUMEN
BACKGROUND: Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells (ECs) present in blood circulation that are involved in neo-vascularization and correction of damaged endothelial sites. Since EPCs from patients with vascular disorders are impaired and inefficient, allogenic sources from adult or cord blood are considered as good alternatives. However, due to the reaction of immune system against allogenic cells which usually lead to their elimination, we focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. TNFα is one of the main activators of EPCs that recognizes two distinct receptors. TNFR1 is expressed ubiquitously and its interaction with TNFα leads to differentiation and apoptosis, whereas, TNFR2 is expressed predominantly on ECs, immune cells and neural cells and is involved in cell survival and proliferation. Interestingly, it has been shown that different immunosuppressive cells express TNFR2 and this is directly related to their immunosuppressive efficiency. However, little is known about immunological profile and function of TNFR2 in EPCs. METHODS: Using different in-vitro combinations, we performed co-cultures of ECs and T cells to investigate the immunological effect of EPCs on T cells. We interrupted in the TNFα/TNFR2 axis either by blocking the receptor using TNFR2 antagonist or blocking the ligand using T cells derived from TNFα KO mice. RESULTS: We demonstrated that EPCs are able to suppress T cell proliferation and modulate them towards less pro-inflammatory and active phenotypes. Moreover, we showed that TNFα/TNFR2 immune-checkpoint pathway is critical in EPC immunomodulatory effect. CONCLUSIONS: Our results reveal for the first time a mechanism that EPCs use to suppress immune cells, therefore, enabling them to form new immunosuppressive vessels. Furthermore, we have shown the importance of TNFα/TNFR2 axis in EPCs as an immune checkpoint pathway. We believe that targeting TNFR2 is especially crucial in cancer immune therapy since it controls two crucial aspects of tumor microenvironment: 1) Immunosuppression and 2) Angiogenesis. Video Abstract. (MP4 46355 kb).
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Células Progenitoras Endoteliales , Terapia de Inmunosupresión , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Linfocitos T/citología , Factor de Necrosis Tumoral alfa/inmunología , Adolescente , Adulto , Anciano , Animales , Células Cultivadas , Técnicas de Cocultivo , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/inmunología , Femenino , Voluntarios Sanos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Transducción de Señal , Adulto JovenRESUMEN
Elevated plasma statured fatty acids (FFAs) cause TLR4/MD2 activation-dependent inflammation and insulin tolerance, which account for the occurrence and development of obesity. It has been confirmed that statured palmitic acid (PA) (the most abundant FFA) could bind MD2 to cause cellular inflammation. The natural compound celastrol could improve obesity, which is suggested via inhibiting inflammation, yet the detailed mechanism for celastrol is still unclear. As celastrol is reported to directly target MD2, we thought disrupting the binding between FFAs and MD2 might be one of the ways for celastrol to inhibit FFAs-caused inflammation and insulin resistance. In this study, we found evidence to support our hypothesis: celastrol could reverse PA-caused TLR4/MD2 activation-dependent insulin resistance, as determined by glucose-lowering ability, cellular glucose uptake, insulin action-related proteins and TLR4/MD2/NF-κB activation. Bioinformatics and cellular experiments showed that both celastrol and PA could bind MD2, and that celastrol could expel PA from cells. Finally, celastrol could reverse high fat diet caused hyperglycemia and obesity, and liver NF-kB activations. Taking together, we proved that celastrol could reverses PA-caused TLR4-MD2 activation-dependent insulin resistance via disrupting PA binding to MD2.
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Resistencia a la Insulina/fisiología , Ácido Palmítico/metabolismo , Receptor Toll-Like 4/efectos de los fármacos , Triterpenos/farmacología , Animales , Dieta Alta en Grasa , Regulación de la Expresión Génica , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Ratones Endogámicos C57BL , Ácido Palmítico/farmacología , Triterpenos Pentacíclicos , Transducción de Señal , Receptor Toll-Like 4/metabolismoRESUMEN
The development of resistance to therapy continues to be a serious clinical problem in lung cancer management. Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is one of the most common chemotherapy drugs to treat non-small-cell lung cancer. However, almost all treatments fail after â¼1 year of treatment because of drug tolerance, probably occurring from the threonine 790 mutation (T790M) of the EGFR, resulting in overactivation of the EGFR. Celastrol is a natural compound that exhibits antiproliferative activity. In this study, we showed that celastrol combined with EGFR-TKIs significantly suppressed cell invasion of lung cancer cells with a T790M mutation by suppressing the EGFR pathway. Combined therapy with celastrol and EGFR-TKIs inhibited tumor growth in vivo. Together, these results suggested that combined therapy with EGFR-TKIs and celastrol may be a more effective treatment of patients with non-small-cell lung cancer with T790M mutations of the EGFR.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Triterpenos/farmacología , Células A549 , Animales , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Triterpenos Pentacíclicos , Inhibidores de Proteínas Quinasas/administración & dosificación , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Triterpenos/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.
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Neoplasias de la Mama/patología , Células Neoplásicas Circulantes/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Análisis Mutacional de ADN , Transición Epitelial-Mesenquimal , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Microscopía Fluorescente , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The dysfunction of endothelial progenitor cells (EPCs) limits their potential for the treatment of ischemia and atherosclerosis. Therefore, we investigated the effect of tripterine on EPC function and examined the underlying mechanisms. The effect of tripterine, an active component of Tripterygium wilfordii Hook, on the enhancement of EPC function and the efficiency of EPC transplantation was investigated in vitro and in vivo. Treatment of EPCs with tripterine at 2.5 µM for 4 h inhibited oxidized low-density lipoprotein (ox-LDL) induced ROS production, cell apoptosis, and cell senescence and improved the migration and tube formation capacities of EPCs treated with ox-LDL (200 µg/ml). In vivo studies showed that tripterine conditioning of EPCs administered to ischemic foci improved blood perfusion and microvascular density in a mouse hindlimb ischemia model. Examination of the underlying mechanisms indicated that the effect of tripterine is mediated by the induction of heat shock protein 32 expression and the inhibition of JNK activation. The present results are of clinical significance because they suggest the potential of tripterine as a therapeutic agent to improve the efficacy of EPC transplantation for the treatment of ischemic diseases.
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Células Progenitoras Endoteliales/metabolismo , Hemo-Oxigenasa 1/metabolismo , Proteínas de la Membrana/metabolismo , Triterpenos/farmacología , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Separación Celular , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Células Progenitoras Endoteliales/efectos de los fármacos , Células Progenitoras Endoteliales/enzimología , Activación Enzimática/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Isquemia/patología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Triterpenos Pentacíclicos , Flujo Sanguíneo Regional/efectos de los fármacos , Factores de TiempoRESUMEN
BACKGROUND/AIMS: Atherosclerosis is associated with dysfunction of endothelial progenitor cells (EPCs). Tripterine, a chemical compound derived from the Chinese medicinal plant Tripterygium wilfordii Hook, displays anti-inflammatory properties in several animal models. We hypothesized that tripterine can improve EPC function and thus the efficiency of EPC transplantation. METHODS AND RESULTS: Tripterine preconditioning (2.5 µM, 4 h) improved EPC proliferation, tube formation, migration, and adhesion, and reduced apoptosis in cells cultured in ox-LDL (200 µg/ml). Tripterine restored integrin-linked kinase (ILK) levels downregulated by ox-LDL in EPCs, suggesting the involvement of the ILK/Akt pathway. Small interfering RNA-mediated depletion of ILK and dominant-negative ILK transduction inhibited the phosphorylation of the ILK downstream signaling targets protein kinase B/Akt and glycogen synthase kinase 3-beta (GSK-3ß), and reduced ß-catenin and cyclin D1 expression. In atherosclerotic mice injected with green fluorescent protein-labeled EPCs to evaluate EPC function, tripterine decreased aortic lesions and plaque deposition, and injection of tripterine-treated EPCs restored ILK levels. CONCLUSION: The present results suggest that tripterine improves vascular function in atherosclerosis by enhancing EPC function through a mechanism involving the ILK signaling pathway.
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Antiinflamatorios/farmacología , Aterosclerosis/terapia , Células Progenitoras Endoteliales/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Triterpenos/farmacología , Animales , Aterosclerosis/metabolismo , Adhesión Celular/efectos de los fármacos , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/trasplante , Regulación de la Expresión Génica/efectos de los fármacos , Lipoproteínas LDL/efectos adversos , Ratones , Triterpenos Pentacíclicos , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
BACKGROUND: Celastrol is a novel anti-tumor agent. Ways to further enhance this effect of celastrol has attracted much research attention. METHODS AND RESULTS: Here, we report that celastrol treatment can elevate miR-223 in human breast cancer cell line MCF-7 and prostate cancer PC3. Down-regulating miR-223 could increase the number of viable cells, yet it further reduced viable cells in samples that were treated by celastrol; up-regulation of miR-223 displayed opposite effects. Celastrol's miR-223 induction might be due to NF-κB inhibition and transient mTOR activation: these two events occurred prior to miR-223 elevation in celastrol-treated cells. NF-κB inhibitor, like celastrol, could induce miR-223; the induction of miR-223 by NF-κB inhibitor or celastrol was reduced by the use of mTOR inhibitor. Finally and interestingly, miR-223 also could affect NF-κB and mTOR and the effects were different between cells treated or not treated with celastrol, thus providing an explanation for differing effects of miR-223 alteration on cellular viability in the presence of celastrol or not. CONCLUSIONS: For the first time, we disclose that celastrol could induce miR-223 in breast and prostate cancer cells, and that inhibiting miR-223 could further reduce the living cells in celastrol-treated cancer cell lines. We thus provide a novel way to increase celastrol's anti-cancer effects.
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Neoplasias de la Mama/genética , MicroARNs/biosíntesis , Neoplasias de la Próstata/genética , Triterpenos/administración & dosificación , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Masculino , MicroARNs/antagonistas & inhibidores , FN-kappa B/antagonistas & inhibidores , Triterpenos Pentacíclicos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Flow cytometry, in conjunction with immunoprecipitation (IP-FCM), is suggested to have some advantages to conventional IP-western blot technology in analyzing protein complexes. In this paper, to further examine its practicability, we test the use of IP-FCM in detecting the HSP90 complex, which has gained importance in drug research and development and involves more than a dozen components. We found that IP-FCM could effectively detect HSP70, p23, Cdc37, and Cdk6 components in the HSP90 complex naturally formed in U937 cells when this complex was captured by anti-HSP90 antibody-coated polystyrene microspheres. IP-FCM could also detect alteration in components caused by treating cells with HSP90 inhibitors. In a cell-free environment, IP-FCM could detect the direct effects of ATP and/or HSP90 inhibitors (17-N-allylamino-17-demethoxygeldanamycin or celastrol) in causing component dissociation and the time- and dose-effects of inhibitor-caused dissociation. IP-FCM is a practical and powerful platform for analyzing HSP90 complex components, and is thus a useful tool in studying HSP90 complex function and screening inhibitors.
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Citometría de Flujo/métodos , Proteínas HSP90 de Choque Térmico/análisis , Inmunoprecipitación/métodos , Western Blotting , Línea Celular Tumoral , Humanos , MicroesferasRESUMEN
BACKGROUND: Celastrol is a promising anti-tumor agent, yet it also elevates heat shock proteins (HSPs), especially HSP70, this effect believed to reduce its anti-tumor effects. Concurrent use of siRNA to increase celastrol's anti-tumor effects through HSP70 interference has been reported, but because siRNA technology is difficult to clinically apply, an alternative way to curb unwanted HSP70 elevation caused by celastrol treatment is worth exploring. METHODS: In this work, we explore three alternative strategies to control HSP70 elevation: (1) Searching for cancer cell types that show no HSP70 elevation in the presence of celastrol (thus recommending themselves as suitable targets); (2) Modifying HSP70-inducing chemical groups, i.e.: the carboxyl group in celastrol; and (3) Using signaling molecule inhibitors to specifically block HSP70 elevation while protecting and/or enhancing anti-tumor effects. RESULTS: The first strategy was unsuccessful since celastrol treatment increased HSP70 in all 7 of the cancer cell types tested, this result related to HSF1 activation. The ubiquity of HSF1 expression in different cancer cells might explain why celastrol has no cell-type limitation for HSP70 induction. The second strategy revealed that modification of celastrol's carboxyl group abolished its ability to elevate HSP70, but also abolished celastrol's tumor inhibition effects. In the third strategy, 11 inhibitors for 10 signaling proteins reportedly related to celastrol action were tested, and five of these could reduce celastrol-caused HSP70 elevation. Among these, the peptide deformylase (PDF) inhibitor, actinonin, could synergize celastrol's proliferation inhibition. CONCLUSIONS: Concurrent use of the chemical agent actinonin could reduce celastrol's HSP70 elevation and also enhance proliferation inhibition by celastrol. This combination presents a novel alternative to siRNA technology and is worth further investigation for its potentially effective anti-tumor action.
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Proteínas HSP70 de Choque Térmico/genética , Triterpenos/farmacología , Amidohidrolasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Triterpenos Pentacíclicos , Fosforilación/efectos de los fármacos , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Circulating tumor cells (CTCs) represent a rare and heterogeneous population of cancer cells that are detached from the tumor site and entered blood or lymphatic circulation. Once disseminated in distant tissues, CTCs could remain dormant or create a tumor mass causing serious danger for patients. Many technologies exist to isolate CTCs from patients' blood samples, mostly based on microfluidic systems or by sorting them according to their surface antigens, notably EpCAM, and/or cytokeratins for carcinoma. ScreenCell has developed an easy-to-use, antigen-independent, rapid, cost-effective, and efficient technology that isolates CTCs according to their bigger size compared to the blood cells. This study provides the technical information necessary to isolate and characterize CTCs from mouse blood. By using blood samples from transgenic mice with breast cancer or from WT mice in which we spiked cancer cells, we showed that ScreenCell technology is compatible with standard EDTA blood collection tubes. Furthermore, the ScreenCell Cyto kit could treat up to 500 µl and the ScreenCell MB kit up to 200 µl of mouse blood. As the ScreenCell MB kit captures unaltered live CTCs, we have shown that their DNA could be efficiently extracted, and the isolated cells could be grown in culture. In conclusion, ScreenCell provides a rapid, easy, antigen-independent, cost-effective, and efficient technology to isolate and characterize CTCs from the blood samples of cancer patients and murine models. Thanks to this technology CTCs could be captured fixed or alive. Murine cancer models are extensively used in pre-clinical studies. Therefore, this study demonstrates the crucial technical points necessary while manipulating mouse blood samples using ScreenCell technology.
Asunto(s)
Separación Celular , Ratones Transgénicos , Células Neoplásicas Circulantes , Células Neoplásicas Circulantes/patología , Células Neoplásicas Circulantes/metabolismo , Animales , Ratones , Separación Celular/métodos , Femenino , Humanos , Línea Celular Tumoral , Neoplasias de la Mama/patología , Neoplasias de la Mama/sangreRESUMEN
Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties.
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Células Endoteliales/metabolismo , Células Madre Mesenquimatosas/fisiología , Neovascularización Fisiológica/fisiología , Receptor Toll-Like 3/fisiología , Animales , Ciclo Celular , División Celular , Movimiento Celular , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Células Endoteliales/citología , Endotelio Vascular/fisiología , Femenino , Sangre Fetal/citología , Regulación de la Expresión Génica/fisiología , Miembro Posterior/irrigación sanguínea , Humanos , Recién Nacido , Isquemia/cirugía , Ligandos , Lipoproteínas LDL/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/antagonistas & inhibidores , MicroARNs/biosíntesis , MicroARNs/genética , Oligonucleótidos/farmacología , Poli I-C/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/biosíntesis , Receptores Toll-Like/agonistas , Receptores Toll-Like/biosíntesis , Receptores Toll-Like/genética , Factor de Necrosis Tumoral alfa/farmacología , Cicatrización de HeridasRESUMEN
A new paradigm has emerged recently, which consists in shifting from cell therapy to a more flexible acellular "extracellular vesicle (EV) therapy" approach, thereby opening a new and promising field in nanomedicine. Important technical limitations have still to be addressed for the large-scale production of clinical-grade EV. Cells are cultured in media supplemented with human platelet lysate (hPL) (xenogenic-free) or GMP-grade fetal calf serum (FCS). However, these additives contain high amounts of EV that cannot be separated from cell-secreted -EV. Therefore, cells are generally maintained in additive-free medium during the EV secretion phase, however this can substantially limit their survival. In the present work, we developed a method to prepare vesicle-free hPL (EV-free hPL) or vesicle-free FCS (EV-free FCS) using tangential flow filtration (TFF). We show a very efficient EV depletion (>98%) for both pure hPL and FCS, with a highly conserved protein content. Culture medium containing our EV-free additives supported the survival of human bone marrow MSC (BM-MSC). MSC could survive at least 216 h, their conditioned medium being collected and changed every 72 h. Both the cell survival and the cumulative EV production were substantially higher than in the starving conditions classically used for EV production. In EV-free hPL containing medium, we show that purified EV kept their morphologic and molecular characteristics throughout the production. Finally, we tested our additives with 3 other cell types, human primary Endothelial Colony Forming Cells (ECFC) and two non-adherent human cell lines, Jurkat and THP-1. We confirmed that both EV-free hPL and FCS were able to maintain cell survival and EV production for at least 216 h. Our method provides therefore a new option to help producing large amounts of EV from virtually any mammalian cells, particularly those that do not tolerate starvation. This method can apply to any animal serum for research and development purpose. Moreover, EV-free hPL is clinical-grade compatible and allows preparing xenobiotic-free media for massive therapeutic EV production in both 2D (cell plates) and 3D (bioreactor) setting.
Asunto(s)
Vesículas Extracelulares , Células Madre Mesenquimatosas , Animales , Humanos , Células Cultivadas , Diferenciación Celular , Proliferación Celular , Plaquetas/metabolismo , Técnicas de Cultivo de Célula , MamíferosRESUMEN
OBJECTIVE: To evaluate the possible merit of endothelial markers for the prediction of ischaemic digital ulcers in patients with systemic sclerosis (SSc). METHODS: Circulating endothelial progenitor cells (EPC), circulating endothelial cells and serum levels of placental growth factor (PlGF), soluble vascular adhesion molecule and vascular endothelial growth factor were measured in a prospective cohort of 100 SSc patients. The primary outcome was the occurrence of one or more new ischaemic digital ulcers during a planned 3-year follow-up. RESULTS: After the follow-up period, 17 patients developed new digital ulcers. By multivariate analysis focused on biomarkers, high PlGF serum levels and low EPC counts were identified as predictors of the occurrence of at least one new digital ulcer. In a secondary model including biomarkers together with clinical SSc characteristics all predictors of digital ulcers defined by p≤0.1 in univariate analysis, high PlGF serum levels (HR 7.26, 95% CI 1.92 to 27.41) and a history of digital ulcers (HR 9.32, 95% CI 1.51 to 59.83) were identified as independent predictors of a new digital ulcer. In an alternative model excluding patients with a history of digital ulcers at baseline, high PlGF serum levels (HR 13.46, 95% CI 1.58 to 114.73) and low EPC counts (HR 7.95, 95% CI 2.09 to 30.09) remained predictive of new digital ulcer occurrence during follow-up. CONCLUSION: This study identified high PlGF serum levels and low circulating EPC counts as predictors of new digital ulcers in SSc. It highlights the critical role of angiogenesis in this vascular outcome. These markers may improve digital ulcer risk stratification and therefore allow earlier therapeutic intervention.