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1.
Cells ; 10(9)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34571827

RESUMO

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.


Assuntos
Proteínas de Choque Térmico/metabolismo , Células Secretoras de Insulina/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/fisiologia , Proteólise , Resposta a Proteínas não Dobradas/fisiologia
2.
Mol Cell Endocrinol ; 477: 39-47, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29792912

RESUMO

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.


Assuntos
Citoproteção , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Prolactina/farmacologia , Substâncias Protetoras/farmacologia , Adulto , Animais , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Citoproteção/efeitos dos fármacos , Células HEK293 , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Chaperonas Moleculares
3.
BMC Cancer ; 17(1): 194, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28298203

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Caspases/metabolismo , Azul de Metileno/administração & dosagem , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Apoptose , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Azul de Metileno/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Transdução de Sinais
4.
Stem Cells Transl Med ; 6(1): 161-173, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28170177

RESUMO

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.


Assuntos
Tecido Adiposo/patologia , Carcinogênese/patologia , Imunomodulação , Células-Tronco Pluripotentes/citologia , Fator de Crescimento Transformador beta1/farmacologia , Animais , Biomarcadores/metabolismo , Carcinogênese/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Camadas Germinativas/citologia , Humanos , Imunomodulação/efeitos dos fármacos , Cariótipo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Células-Tronco Pluripotentes/transplante , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Baço/citologia , Estresse Fisiológico , Teratoma/patologia
5.
Cell Transplant ; 24(2): 247-61, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-24256689

RESUMO

Pancreatic islet microencapsulation constitutes an attractive therapy for type 1 diabetes mellitus; however, long-term ß-cell function remains a major problem. Loss of extracellular matrix interactions during islet isolation dramatically affects ß-cell viability. We have previously shown beneficial effects of laminin (LN) in human islet cultures. Herein, we investigated whether LN could improve the outcome of transplantation after islet microencapsulation in Biodritin, an alginate-based material. To test LN-Biodritin stability, microcapsules were subjected to different types of in vitro stress. Focusing on biocompatibility, empty microcapsules were coincubated with the RAW 264.7 macrophage cell line for up to 24 h, and empty beads were implanted IP in mice and retrieved for analyses after 7 and 30 days. Upon culturing for 48 h, mRNA, protein levels, and caspase 3 activity were evaluated in islets microencapsulated with LN-Biodritin. Mice rendered diabetic by streptozotocin injection were transplanted with microencapsulated islets, followed by assessment of body weight, glycemia, and graft function (evaluated by OGTT). Graft efficiency was observed upon microencapsulated islet explantation. The results obtained showed that LN-Biodritin microcapsules were as stable and biocompatible as Biodritin. Modulation of mRNA and protein levels suggested protection against apoptosis and islet stress. Mice transplanted with LN-Biodritin microencapsulated islets presented a better outcome at 198 days postsurgery. Graft explantation led animals to hyperglycemia. In conclusion, LN-Biodritin constitutes a very promising biomaterial for islet transplantation.


Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Cápsulas/química , Sulfatos de Condroitina/química , Diabetes Mellitus Experimental/terapia , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/citologia , Laminina/química , Animais , Caspase 3/genética , Caspase 3/metabolismo , Células Cultivadas , Inseminação Artificial Heteróloga , Insulina/metabolismo , Secreção de Insulina , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transcriptoma , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
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