RESUMEN

Pancreatic ductal adenocarcinomas (PDAC) are the fourth leading cause of death due to neoplasms. In view of the urgent need of effective treatments for PDAC, photodynamic therapy (PDT) appears as a promising alternative. However, its efficacy against PDAC and the mechanisms involved in cell death induction remain unclear. In this study, we set out to evaluate PDT's cytotoxicity using methylene blue (MB) as a photosensitizer (PS) (MB-PDT) and to evaluate the contribution of necroptosis in its effect in human PDAC cells. Our results demonstrated that MB-PDT induced significant death of different human PDAC models presenting two different susceptibility profiles. This effect was independent of MB uptake or its subcellular localization. We found that the ability of triggering necroptosis was determinant to increase the treatment efficiency. Analysis of single cell RNA-seq data from normal and neoplastic human pancreatic tissues showed that specific necroptosis proteins RIPK1, RIPK3 and MLKL presented significant higher expression levels in cells displaying a transformed phenotype providing further support to the use of approaches that activate necroptosis, like MB-PDT, as useful adjunct to surgery of PDAC to tackle the problem of microscopic residual disease as well as to minimize the chance of local and metastatic recurrence.

Asunto(s)

Adenocarcinoma , Fotoquimioterapia , Humanos , Azul de Metileno/farmacología , Necroptosis , Fármacos Fotosensibilizantes/farmacología , Fotoquimioterapia/métodos , Apoptosis , Neoplasias Pancreáticas

RESUMEN

During type 1 diabetes mellitus (T1DM) development, beta-cells undergo intense endoplasmic reticulum (ER) stress that could result in apoptosis through the failure of adaptation to the unfolded protein response (UPR). Islet transplantation is considered an attractive alternative among beta-cell replacement therapies for T1DM. To avoid the loss of beta-cells that will jeopardize the transplant's outcome, several strategies are being studied. We have previously shown that prolactin induces protection against proinflammatory cytokines and redox imbalance-induced beta-cell death by increasing heat-shock protein B1 (HSPB1) levels. Since the role of HSPB1 in beta cells has not been deeply studied, we investigated the mechanisms involved in unbalanced protein homeostasis caused by intense ER stress and overload of the proteasomal protein degradation pathway. We tested whether HSPB1-mediated cytoprotective effects involved UPR modulation and improvement of protein degradation via the ubiquitin-proteasome system. We demonstrated that increased levels of HSPB1 attenuated levels of pro-apoptotic proteins such as CHOP and BIM, as well as increased protein ubiquitination and the speed of proteasomal protein degradation. Our data showed that HSPB1 induced resistance to proteotoxic stress and, thus, enhanced cell survival via an increase in beta-cell proteolytic capacity. These results could contribute to generate strategies aimed at the optimization of beta-cell replacement therapies.

Asunto(s)

Proteínas de Choque Térmico/metabolismo , Células Secretoras de Insulina/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Apoptosis/fisiología , Muerte Celular/fisiología , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Oxidación-Reducción , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/fisiología , Proteolisis , Respuesta de Proteína Desplegada/fisiología

RESUMEN

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. SIGNIFICANCE: We describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes.

Asunto(s)

Glicopéptidos , Glicoproteínas , Animales , Bovinos , Glicoproteínas/metabolismo , Glicosilación , Oligosacáridos , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polisacáridos

RESUMEN

Beta-cell death and dysfunction are involved in the development of type 1 and 2 diabetes. ER-stress impairs beta-cells function resulting in pro-apoptotic stimuli that promote cell death. Hence, the identification of protective mechanisms in response to ER-stress could lead to novel therapeutic targets and insight in the pathology of these diseases. Here, we report the identification of proteins involved in dysregulated pathways upon thapsigargin treatment of MIN6 cells. Utilizing quantitative proteomics we identified upregulation of proteins involved in protein folding, unfolded protein response, redox homeostasis, proteasome processes associated with endoplasmic reticulum and downregulation of TCA cycle, cellular respiration, lipid metabolism and ribosome assembly processes associated to mitochondria and eukaryotic initiation translation factor components. Subsequently, pro-inflammatory cytokine treatment was performed to mimic pathological changes observed in beta-cells during diabetes. Cytokines induced ER stress and impaired mitochondrial function in beta-cells corroborating the results obtained with the proteomic approach. HSPB1 levels are increased by prolactin on pancreatic beta-cells and this protein is a key factor for cytoprotection although its role has not been fully elucidated. Here we show that while up-regulation of HSPB1 was able to restore the mitochondrial dysfunction induced by beta-cells' exposure to inflammatory cytokines, silencing of this chaperone abrogated the beneficial effects promoted by PRL. Taken together, our results outline the importance of HSPB1 to mitigate beta-cell dysfunction. Further studies are needed to elucidate its role in diabetes.

Asunto(s)

Estrés del Retículo Endoplásmico/fisiología , Proteínas de Choque Térmico/metabolismo , Mitocondrias/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Respiración de la Célula/fisiología , Citocinas/metabolismo , Diabetes Mellitus/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/fisiología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiología , Ratones , Mitocondrias/fisiología , Chaperonas Moleculares/fisiología , Proteómica/métodos , Tapsigargina/farmacología

RESUMEN

Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

Asunto(s)

Neoplasias de la Mama/patología , Luz , Antioxidantes/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Carcinogénesis/efectos de la radiación , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Femenino , Ferroptosis/efectos de los fármacos , Ferroptosis/efectos de la radiación , Humanos , Lípidos/química , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de la radiación , Azul de Metileno/farmacología , Azul de Metileno/uso terapéutico , Modelos Biológicos , Necroptosis/efectos de los fármacos , Necroptosis/efectos de la radiación , Oxidación-Reducción , Fotoquimioterapia , Neoplasias de la Mama Triple Negativas/patología

RESUMEN

Placental malaria (PM) is associated with severe inflammation leading to abortion, preterm delivery, and intrauterine growth restriction. Innate immunity responses play critical roles, but the mechanisms underlying placental immunopathology are still unclear. Here, we investigated the role of inflammasome activation in PM by scrutinizing human placenta samples from an endemic area and ablating inflammasome components in a PM mouse model. The reduction in birth weight in babies from infected mothers is paralleled by increased placental expression of AIM2 and NLRP3 inflammasomes. Using genetic dissection, we reveal that inflammasome activation pathways are involved in the production and detrimental action of interleukin-1ß (IL-1ß) in the infected placenta. The IL-1R pharmacological antagonist Anakinra improved pregnancy outcomes by restoring fetal growth and reducing resorption in an experimental model. These findings unveil that IL-1ß-mediated signaling is a determinant of PM pathogenesis, suggesting that IL-1R antagonists can improve clinical outcomes of malaria infection in pregnancy.

Asunto(s)

Inflamasomas/efectos de los fármacos , Interleucina-1beta/inmunología , Malaria Falciparum/inmunología , Malaria/inmunología , Plasmodium falciparum/patogenicidad , Complicaciones Parasitarias del Embarazo/inmunología , Transducción de Señal/efectos de los fármacos , Animales , Caspasa 1/genética , Caspasa 1/inmunología , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Femenino , Regulación de la Expresión Génica , Humanos , Inmunidad Innata , Factores Inmunológicos/farmacología , Inflamasomas/genética , Inflamasomas/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Malaria/tratamiento farmacológico , Malaria/genética , Malaria/parasitología , Malaria Falciparum/genética , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Plasmodium berghei/inmunología , Plasmodium berghei/patogenicidad , Plasmodium falciparum/inmunología , Embarazo , Complicaciones Parasitarias del Embarazo/genética , Complicaciones Parasitarias del Embarazo/parasitología , Complicaciones Parasitarias del Embarazo/prevención & control , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/inmunología , Transducción de Señal/inmunología , Células THP-1 , Trofoblastos/efectos de los fármacos , Trofoblastos/inmunología , Trofoblastos/parasitología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

Photodynamic therapy (PDT) appears as a promising alternative in the treatment of breast cancer since it can be highly effective in curing cancer while preserving normal tissue. However, predicting outcomes in PDT still constitutes a great challenge. One of the parameters that are usually empirically determined is the rate of photon flux delivered to the tissue (light fluence rate). In the present study, we intended to understand why monolayers of human cells derived from mammary adenocarcinomas (MDA-MB-231 and MCF-7) respond quite differently to fluence rates (cells were irradiated either for 6 or for 16 min) at a fixed light dose (4.5 J cm-2 ) delivered with an array of LEDs in a typical methylene blue PDT protocol. While death rates of MDA-MB-231 cells were insensitive to the fluence rate, MCF-7 cells showed a quite impressive (three times) decrease in cell death levels in the shorter irradiation protocol. Independent on cell type cell death was invariably correlated with the depletion of reduced glutathione intracellular levels and consequently with widespread redox misbalance. Our data show the potential to optimize fluence rates to provide exhaustion of the cell antioxidant responses in order to circumvent therapy resistance of breast tumors.

Asunto(s)

Neoplasias de la Mama/patología , Glutatión/metabolismo , Azul de Metileno/farmacología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Estrés Oxidativo

RESUMEN

Malaria in pregnancy is a public health concern in malaria-endemic areas. Accumulation of maternal immune cells in the placenta and increased levels of inflammatory cytokines caused by sequestration of Plasmodium falciparum-infected erythrocytes have been associated to poor neonatal outcomes, including low birth weight because of fetal growth restriction. Little is known about the molecular changes occurring in a P. falciparum-infected placenta that has developed placental malaria during pregnancy but had the parasites cleared by pharmacological treatment (past infection). We conducted an integrated proteome, phosphoproteome and glycoproteome analysis in past P. falciparum-infected placentas aiming to find molecular changes associated with placental malaria. A total of 2946 proteins, 1733 N-linked glycosites and 4100 phosphosites were identified and quantified in this study, disclosing overrepresented processes related to oxidative stress, protein folding and regulation of apoptosis in past-infected placentas Moreover, AKT and ERK signaling pathways activation, together with clinical data, were further correlated to an increased apoptosis in past-infected placentas. This study showed apoptosis-related mechanisms associated with placental malaria that can be further explored as therapeutic target against adverse pregnancy outcomes.

Asunto(s)

Malaria Falciparum/metabolismo , Placenta/metabolismo , Complicaciones Parasitarias del Embarazo/metabolismo , Proteómica/métodos , Animales , Modelos Animales de Enfermedad , Femenino , Glicosilación , Humanos , Sistema de Señalización de MAP Quinasas , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Ratones , Fosforilación , Placenta/parasitología , Embarazo , Complicaciones Parasitarias del Embarazo/tratamiento farmacológico , Mapas de Interacción de Proteínas

RESUMEN

The success of islet transplantation has improved lately. Unfortunately, it is still compromised by cell loss. We have shown that prolactin (PRL) inhibits beta-cell apoptosis and up-regulates the antiapoptotic Heat Shock Protein B1 (HSPB1) in human islets. Since its function in pancreatic islets has not been studied, we explored the role of HSPB1 in PRL-induced beta-cell survival. The significant PRL-induced cytoprotection in control cells was abrogated in HSPB1 silenced cells, overexpression of HSPB1 recovered survival. PRL-mediated inhibition of cytokine-induced caspase activities and cytokine-induced decrease of BCL-2/BAX ratio was significantly reverted in knocked-down cells. Kinetics of HSPB1 and HSF1 expression were studied in primary cultures of murine and human pancreatic islets. These findings are highly relevant for the improvement of clinical islet transplantation success rate since our results demonstrated a key role for HSPB1 pointing it as a promising target for beta-cell cytoprotection through the up-regulation of an endogenous protective pathway.

Asunto(s)

Citoprotección , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Prolactina/farmacología , Sustancias Protectoras/farmacología , Adulto , Animales , Apoptosis/efectos de los fármacos , Citocinas/metabolismo , Citoprotección/efectos de los fármacos , Células HEK293 , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Chaperonas Moleculares

RESUMEN

BACKGROUND: Breast cancer is the main cause of mortality among women. The disease presents high recurrence mainly due to incomplete efficacy of primary treatment in killing all cancer cells. Photodynamic therapy (PDT), an approach that causes tissue destruction by visible light in the presence of a photosensitizer (Ps) and oxygen, appears as a promising alternative therapy that could be used adjunct to chemotherapy and surgery for curing cancer. However, the efficacy of PDT to treat breast tumours as well as the molecular mechanisms that lead to cell death remain unclear. METHODS: In this study, we assessed the cell-killing potential of PDT using methylene blue (MB-PDT) in three breast epithelial cell lines that represent non-malignant conditions and different molecular subtypes of breast tumours. Cells were incubated in the absence or presence of MB and irradiated or not at 640 nm with 4.5 J/cm2. We used a combination of imaging and biochemistry approaches to assess the involvement of classical autophagic and apoptotic pathways in mediating the cell-deletion induced by MB-PDT. The role of these pathways was investigated using specific inhibitors, activators and gene silencing. RESULTS: We observed that MB-PDT differentially induces massive cell death of tumour cells. Non-malignant cells were significantly more resistant to the therapy compared to malignant cells. Morphological and biochemical analysis of dying cells pointed to alternative mechanisms rather than classical apoptosis. MB-PDT-induced autophagy modulated cell viability depending on the cell model used. However, impairment of one of these pathways did not prevent the fatal destination of MB-PDT treated cells. Additionally, when using a physiological 3D culture model that recapitulates relevant features of normal and tumorous breast tissue morphology, we found that MB-PDT differential action in killing tumour cells was even higher than what was detected in 2D cultures. CONCLUSIONS: Finally, our observations underscore the potential of MB-PDT as a highly efficient strategy which could use as a powerful adjunct therapy to surgery of breast tumours, and possibly other types of tumours, to safely increase the eradication rate of microscopic residual disease and thus minimizing the chance of both local and metastatic recurrence.

Asunto(s)

Neoplasias de la Mama/metabolismo , Caspasas/metabolismo , Azul de Metileno/administración & dosificación , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/administración & dosificación , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Azul de Metileno/farmacología , Fármacos Fotosensibilizantes/farmacología , Transducción de Señal

RESUMEN

Adult mesenchymal stromal cell-based interventions have shown promising results in a broad range of diseases. However, their use has faced limited effectiveness owing to the low survival rates and susceptibility to environmental stress on transplantation. We describe the cellular and molecular characteristics of multilineage-differentiating stress-enduring (Muse) cells derived from adipose tissue (AT), a subpopulation of pluripotent stem cells isolated from human lipoaspirates. Muse-AT cells were efficiently obtained using a simple, fast, and affordable procedure, avoiding cell sorting and genetic manipulation methods. Muse-AT cells isolated under severe cellular stress, expressed pluripotency stem cell markers and spontaneously differentiated into the three germ lineages. Muse-AT cells grown as spheroids have a limited proliferation rate, a diameter of ∼15 µm, and ultrastructural organization similar to that of embryonic stem cells. Muse-AT cells evidenced high stage-specific embryonic antigen-3 (SSEA-3) expression (∼60% of cells) after 7-10 days growing in suspension and did not form teratomas when injected into immunodeficient mice. SSEA-3+ -Muse-AT cells expressed CD105, CD29, CD73, human leukocyte antigen (HLA) class I, CD44, and CD90 and low levels of HLA class II, CD45, and CD34. Using lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays, we have shown that Muse-AT cells have anti-inflammatory activities downregulating the secretion of proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. Muse-AT cells spontaneously gained transforming growth factor-ß1 expression that, in a phosphorylated SMAD2-dependent manner, might prove pivotal in their observed immunoregulatory activity through decreased expression of T-box transcription factor in T cells. Collectively, the present study has demonstrated the feasibility and efficiency of obtaining Muse-AT cells that can potentially be harnessed as immunoregulators to treat immune-related disorders. Stem Cells Translational Medicine 2017;6:161-173.

Asunto(s)

Tejido Adiposo/patología , Carcinogénesis/patología , Inmunomodulación , Células Madre Pluripotentes/citología , Factor de Crecimiento Transformador beta1/farmacología , Animales , Biomarcadores/metabolismo , Carcinogénesis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Estratos Germinativos/citología , Humanos , Inmunomodulación/efectos de los fármacos , Cariotipo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Células Madre Pluripotentes/trasplante , Células RAW 264.7 , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Bazo/citología , Estrés Fisiológico , Teratoma/patología

RESUMEN

Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits.Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/ß-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1.Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis.

Asunto(s)

Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Glipicanos/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Glipicanos/metabolismo , Humanos , Células MCF-7 , Ratones Desnudos , Interferencia de ARN , Trasplante Heterólogo , Carga Tumoral/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo

RESUMEN

BACKGROUND: The REversion-inducing Cysteine-rich protein with Kazal motif (RECK) is a well-known inhibitor of matrix metalloproteinases (MMPs) and cellular invasion. Although high expression levels of RECK have already been correlated with a better clinical outcome for several tumor types, its main function, as well as its potential prognostic value for breast cancer patients, remain unclear. METHODS: The RECK expression profile was investigated in a panel of human breast cell lines with distinct aggressiveness potential. RECK functional analysis was undertaken using RNA interference methodology. RECK protein levels were also analyzed in 1040 cases of breast cancer using immunohistochemistry and tissue microarrays (TMAs). The association between RECK expression and different clinico-pathological parameters, as well as the overall (OS) and disease-free (DFS) survival rates, were evaluated. RESULTS: Higher RECK protein expression levels were detected in more aggressive breast cancer cell lines (T4-2, MDA-MB-231 and Hs578T) than in non-invasive (MCF-7 and T47D) and non-tumorigenic (S1) cell lines. Indeed, silencing RECK in MDA-MB-231 cells resulted in elevated levels of pro-MMP-9 and increased invasion compared with scrambled (control) cells, without any effect on cell proliferation. Surprisingly, by RECK immunoreactivity analysis on TMAs, we found no association between RECK positivity and survival (OS and DFS) in breast cancer patients. Even considering the different tumor subtypes (luminal A, luminal B, Her2 type and basal-like) or lymph node status, RECK remained ineffective for predicting the disease outcome. Moreover, by multivariate Cox regression analysis, we found that RECK has no prognostic impact for OS and DFS, relative to standard clinical variables. CONCLUSIONS: Although it continues to serve as an invasion and MMP inhibitor in breast cancer, RECK expression analysis is not useful for prognosis of these patients.

Asunto(s)

Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Proteínas Ligadas a GPI/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Femenino , Proteínas Ligadas a GPI/genética , Expresión Génica , Humanos , Estimación de Kaplan-Meier , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Clasificación del Tumor , Pronóstico , Modelos de Riesgos Proporcionales , Factores de Riesgo , Carga Tumoral

RESUMEN

In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them to those of primary beta-cells using DIGE followed by MS. The results were validated by Western blotting. An average of 1800 spots was detected with less than 1% exhibiting differential abundance. Proteins more abundant in human islets, such as Caldesmon, are involved in the regulation of cell contractility, adhesion dependent signaling, and cytoskeletal organization. In contrast, almost all proteins more abundant in insulinoma cells, such as MAGE2, were first described here and could be related to cell survival and resistance to chemotherapy. Our proteomic data provides, for the first time, a molecular snapshot of the orchestrated changes in expression of proteins involved in key processes which could be correlated with the altered phenotype of human beta-cells. Collectively our observations prompt research towards the establishment of bioengineered human beta-cells providing a new and needed source of cultured human beta-cells for beta-cell research, along with the development of new therapeutic strategies for detection, characterization and treatment of insulinomas.

Asunto(s)

Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Proteoma/metabolismo , Adulto , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Insulinoma/genética , Antígenos Específicos del Melanoma/metabolismo , Persona de Mediana Edad , Cultivo Primario de Células , Proteoma/genética , Técnicas de Cultivo de Tejidos , Células Tumorales Cultivadas , Electroforesis Bidimensional Diferencial en Gel

RESUMEN

AIMS: NADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human ß-cell function were investigated. MAIN METHODS AND KEY FINDINGS: In isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22(PHOX), p47(PHOX) and p67(PHOX)), Western blotting (p47(PHOX) and p67(PHOX)) and immunohistochemistry (p47(PHOX), p67(PHOX) and gp91(PHOX)). Immunohistochemistry experiments showed co-localization of p47(PHOX), p67(PHOX) and gp91(PHOX) (isoform 2 of NADPH oxidase-NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. SIGNIFICANCE: These findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human ß-cell function.

Asunto(s)

Islotes Pancreáticos/enzimología , NADPH Oxidasas/metabolismo , Adulto , Secuencia de Bases , Western Blotting , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Humanos , Inmunohistoquímica , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Persona de Mediana Edad , NADPH Oxidasas/antagonistas & inhibidores , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

BACKGROUND: Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-ß1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models. METHODS: The mRNA expression levels of TGF-ß isoforms and their receptors were analyzed by qRT-PCR in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. The highly invasive MDA-MB-231 cell line was treated with different concentrations of recombinant TGF-ß1 and also with pharmacological inhibitors of p38 MAPK and ERK1/2. The migratory and invasive potential of these treated cells were examined in vitro by transwell assays. RESULTS: In general, TGF-ß2, TßRI and TßRII are over-expressed in more aggressive cells, except for TßRI, which was also highly expressed in ZR-75-1 cells. In addition, TGF-ß1-treated MDA-MB-231 cells presented significantly increased mRNA expression of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-ß1 also increased TIMP-2, MMP-2 and MMP-9 protein levels but downregulated RECK expression. Furthermore, we analyzed the involvement of p38 MAPK and ERK1/2, representing two well established Smad-independent pathways, in the proposed mechanism. Inhibition of p38MAPK blocked TGF-ß1-increased mRNA expression of all MMPs and MMP inhibitors analyzed, and prevented TGF-ß1 upregulation of TIMP-2 and MMP-2 proteins. Moreover, ERK1/2 inhibition increased RECK and prevented the TGF-ß1 induction of pro-MMP-9 and TIMP-2 proteins. TGF-ß1-enhanced migration and invasion capacities were blocked by p38MAPK, ERK1/2 and MMP inhibitors. CONCLUSION: Altogether, our results support that TGF-ß1 modulates the mRNA and protein levels of MMPs (MMP-2 and MMP-9) as much as their inhibitors (TIMP-2 and RECK). Therefore, this cytokine plays a crucial role in breast cancer progression by modulating key elements of ECM homeostasis control. Thus, although the complexity of this signaling network, TGF-ß1 still remains a promising target for breast cancer treatment.

Asunto(s)

Neoplasias de la Mama/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Western Blotting , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Homeostasis/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/farmacología

RESUMEN

BACKGROUND: Many studies have evaluated whether there are characteristics related to pancreas donors and the islet isolation process that can influence pancreatic islet yield. However, this analysis has not yet been performed in Brazil, one of the world leaders in whole pancreas organ transplantation (WOPT), where pancreas allocation for pancreatic islet transplantation (PIT) has no officially defined criteria. Definition of parameters that would predict the outcome of islet isolation from local pancreas donors would be useful for defining allocation priority in Brazil. OBJECTIVE: To analyze the relationship between multiple donor-related and islet isolation variables with the total number of isolated pancreatic islet equivalents (IEQ) in a brazilian sample of pancreas donors. METHODS: Several variables were analyzed in 74 pancreata relative to the outcome of total IEQs obtained at the end of the process. RESULTS: In univariate analysis, body mass index (BMI) (p = 0.003), the presence of fatty infiltrates in the pancreas as observed during harvesting (p = 0.042) and pancreas digestion time (p = 0.046) were identified as variables related to a greater IEQ yield. In a multivariate analysis a statistically significant contribution to the variability of islet yield was found only for the BMI (p = 0.017). A ROC curve defined a BMI = 30 as a cut-off point, with pancreata from donors with BMI > 30 yielding more islets than donors with BMI < 30 (p< 0.001). CONCLUSION: These data reinforce the importance of the donor BMI as a defining parameter for successful islet isolation and establishes this variable as a potential pancreas allocation criterion in Brazil, where there is unequal competition for good quality organs between WOPT and PIT.

Asunto(s)

Trasplante de Islotes Pancreáticos/métodos , Trasplante de Páncreas/métodos , Obtención de Tejidos y Órganos/métodos , Adulto , Índice de Masa Corporal , Brasil , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Curva ROC , Sensibilidad y Especificidad , Estadísticas no Paramétricas

RESUMEN

Islet transplant is an innovative treatment for type 1 diabetic patients, which still lies between experimental and approved transplant therapy. Islet cells are seeded in a non-physiological territory where an uncertain fraction will be able to adapt and survive. Thus, the challenge lies in improving the whole procedure, employing the tools of cell biology, immunology and laboratory techniques, in order to reach the results obtained with whole organ transplant. This review describes the procedure, its progress to the present methodology and clinical results obtained. Future perspectives of islet transplantation in the light of recent biotechnological advances are also focused.

Asunto(s)

Diabetes Mellitus Tipo 1/cirugía , Trasplante de Islotes Pancreáticos , Humanos , Trasplante de Islotes Pancreáticos/efectos adversos , Trasplante de Islotes Pancreáticos/métodos , Trasplante de Islotes Pancreáticos/tendencias

RESUMEN

O transplante de ilhotas é um procedimento em desenvolvimento, como alternativa para o tratamento do diabetes tipo 1 que está na fronteira entre o experimental e o clínico. É uma terapia celular na qual as células são implantadas em território diferente do fisiológico em que apenas determinado número incerto conseguirá se adaptar. Aperfeiçoar este processo para obter os mesmos resultados que no transplante de pâncreas, representa um desafio para o qual convergem contribuições da biologia celular, da imunologia e das técnicas de laboratório que se entrelaçam de maneira extremamente complexa. Este trabalho revisa a literatura expondo a evolução do procedimento, a sua metodologia atual e os resultados clínicos obtidos. As perspectivas futuras do transplante diante dos recentes avanços também são discutidas.

---

Islet transplant is an innovative treatment for type 1 diabetic patients, which still lies between experimental and approved transplant therapy. Islet cells are seeded in a non-physiological territory where an uncertain fraction will be able to adapt and survive. Thus, the challenge lies in improving the whole procedure, employing the tools of cell biology, immunology and laboratory techniques, in order to reach the results obtained with whole organ transplant. This review describes the procedure, its progress to the present methodology and clinical results obtained. Future perspectives of islet transplantation in the light of recent biotechnological advances are also focused.

Asunto(s)

Humanos , Diabetes Mellitus Tipo 1/cirugía , Trasplante de Islotes Pancreáticos , Trasplante de Islotes Pancreáticos/efectos adversos , Trasplante de Islotes Pancreáticos/métodos , Trasplante de Islotes Pancreáticos/tendencias