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For a drug candidate to be fully developed takes years and investment of hundreds of millions of dollars. There is no doubt that drug development is difficult and risky, but vital to protecting against devastating disease. This difficulty is clearly evident in BRCA1 and BRCA2 related breast cancer, with current treatment options largely confined to invasive surgical procedures, as well as chemotherapy and radiotherapy regimes which damage healthy tissue and can leave remnant disease. Consequently, patient survival and relapse rates are far from ideal, and new candidate treatments are needed. The preclinical stages of drug discovery are crucial to get right for translation to hospital beds. Disease models must take advantage of current technologies and be accurate for rapid and translatable treatments. Careful selection of cell lines must be coupled with high throughput techniques, with promising results trialled further in highly accurate humanised patient derived xenograft models. Traditional adherent drug screening should transition to 3D culture systems amenable to high throughput techniques if the gap between in vitro and in vivo studies is to be partially bridged. The possibility of organoid, induced pluripotent stem cell, and conditionally reprogrammed in vitro models is tantalising, however protocols are yet to be fully established. This review of BRCA1 and BRCA2 cancer biology and current modelling systems will hopefully guide the design of future drug discovery endeavours and highlight areas requiring improvement.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/tratamento farmacológico , Predisposição Genética para Doença , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Descoberta de Drogas , Feminino , Humanos , MutaçãoRESUMO
OBJECTIVE: Sweet syndrome (SS) is characterized by an inflammatory rash that has been associated with a number of drugs and malignant, inflammatory, and infectious conditions. Rare accounts of Hashimoto thyroiditis (HT) presenting with SS exist in the literature. HT is usually identified after the onset of skin lesions and without signs of overt thyroid dysfunction, and the stage of thyroid disease stage at presentation is variable. METHODS: A search of the PubMed database was performed using search criteria involving combinations of "Sweet syndrome" and "Hashimoto thyroiditis," "autoimmune thyroiditis," or "thyroiditis," and the search was filtered for clinical case reports. Five case reports were identified to describe the coexistence of Sweet syndrome and Hashimoto thyroiditis, and full-text versions of these reports were obtained and reviewed. Of note, cases involving subacute or other types of thyroiditis were excluded. RESULTS: A 57-year-old man presented with painful eruptions on his hands; he was initially treated with antibiotics for presumed cellulitis without relief. Skin biopsy later confirmed SS and subsequent workup identified underlying HT with an elevated thyroid-stimulating hormone of 19.24 mU/L (normal, 0.30 to 4.30 mU/L) and positive thyroid peroxidase (TPO) antibody at 236.4 IU/mL. CONCLUSION: Thyroid function tests should be universally evaluated in the workup of SS, and it may be appropriate to test for TPO antibodies even in the absence of objective thyroid dysfunction. Both SS and HT show immune diathesis, so further work should be undertaken to establish whether a common immunologic trigger exists.
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Recent advances in human pluripotent stem cell (hPSC) research have uncovered different subpopulations within stem cell cultures and have captured a range of pluripotent states that hold distinct molecular and functional properties. At the two ends of the pluripotency spectrum are naïve and primed hPSC, whereby naïve hPSC grown in stringent conditions recapitulate features of the preimplantation human embryo, and the conventionally grown primed hPSC align closer to the early postimplantation embryo. Investigating these cell types will help to define the mechanisms that control early development and should provide new insights into stem cell properties such as cell identity, differentiation and reprogramming. Monitoring cell surface marker expression provides a valuable approach to resolve complex cell populations, to directly compare between cell types, and to isolate viable cells for functional experiments. This review discusses the discovery and applications of cell surface markers to study human pluripotent cell types with a particular focus on the transitions between naïve and primed states. Highlighted areas for future study include the potential functions for the identified cell surface proteins in pluripotency, the production of new high-quality monoclonal antibodies to naïve-specific protein epitopes and the use of cell surface markers to characterise subpopulations within pluripotent states.
Assuntos
Biomarcadores/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/metabolismo , HumanosRESUMO
The MYC and RAS oncogenes are sufficient for transformation of normal rodent cells. This cooperativity is at least in part based on suppression of RAS-induced cellular senescence by MYC and block of MYC-induced apoptosis by RAS - thereby canceling out two main barriers against tumor development. However, it remains unclear whether MYC and RAS cooperate in this way in human cells, where MYC and RAS are not sufficient for transformation. To address this question, we established a combined Tet-inducible H-RASV12 and hydroxytamoxifen-inducible MycER system in normal human BJ fibroblasts. We show here that activation of RAS alone induced senescence while activation of MYC alone or together with RAS triggered DNA damage, induction of p53 and massive apoptosis, suggesting that RAS cannot rescue MYC-induced apoptosis in this system. Although coexpression with MYC reduced certain RAS-induced senescence markers (histone H3 lysine 9 trimethylation and senescence-associated ß-GAL activity), the induction of the senescence marker p16INK4A was further enhanced and the culture ceased to proliferate within a few days, revealing that MYC could not fully suppress RAS-induced senescence. Furthermore, depletion of p53, which enhanced proliferation and rescued the cells from RAS-induced senescence, did not abrogate MYC-induced apoptosis. We conclude that MYC and RAS are unable to cooperate in overcoming senescence and apoptosis in normal human fibroblasts even after depletion of p53, indicating that additional oncogenic events are required to abrogate these fail-safe mechanisms and pave the way for cellular transformation. These findings have implications for our understanding of the transformation process in human cells. Abbreviations and acronyms: CDK: Cyclin-dependent kinase; DDR: DNA damage response; DOX: Doxycycline; EdU: 5-ethynyl-2'-deoxyuridine; FACS: Fluorescence Activated Cell Sorting; MycER: MYC-estrogen receptor; OHT: 4-hydroxytamoxifen; OIS: Oncogene-induced senescence; PP2A: Protein phosphatase 2A; ROS: Reactive oxygen species; SA-ß-GAL: Senescence-associated ß-galactosidase; SAHF: Senescence-associated heterochromatin foci; shRNA: Short hairpin RNA; YFP: Yellow fluorescent protein.
Assuntos
Apoptose , Senescência Celular , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas ras/metabolismo , Apoptose/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Tamoxifeno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas ras/genéticaRESUMO
MYC is a key player in tumor development, but unfortunately no specific MYC-targeting drugs are clinically available. MYC is strictly dependent on heterodimerization with MAX for transcription activation. Aiming at targeting this interaction, we identified MYCMI-6 in a cell-based protein interaction screen for small inhibitory molecules. MYCMI-6 exhibits strong selective inhibition of MYC:MAX interaction in cells and in vitro at single-digit micromolar concentrations, as validated by split Gaussia luciferase, in situ proximity ligation, microscale thermophoresis and surface plasmon resonance (SPR) assays. Further, MYCMI-6 blocks MYC-driven transcription and binds selectively to the MYC bHLHZip domain with a KD of 1.6 ± 0.5 µM as demonstrated by SPR. MYCMI-6 inhibits tumor cell growth in a MYC-dependent manner with IC50 concentrations as low as 0.5 µM, while sparing normal cells. The response to MYCMI-6 correlates with MYC expression based on data from 60 human tumor cell lines and is abrogated by MYC depletion. Further, it inhibits MYC:MAX interaction, reduces proliferation and induces massive apoptosis in tumor tissue from a MYC-driven xenograft tumor model without severe side effects. Since MYCMI-6 does not affect MYC expression, it is a unique molecular tool to specifically target MYC:MAX pharmacologically and it has good potential for drug development.
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Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diaminas/farmacologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Animais , Apoptose/fisiologia , Células COS , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Ativação Transcricional , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Synthetic human pluripotent stem cell (hPSC) culture surfaces with defined physical and chemical properties will facilitate improved research and therapeutic applications of hPSCs. In this study, synthetic surfaces for hPSC culture in E8 medium were produced for screening by modifying two polymer brush coatings [poly(acrylamide-co-acrylic acid) (PAAA) and poly(acrylamide-co-propargyl acrylamide) (PAPA)] to present single peptides. Adhesion of hPSC colonies was more consistently observed on surfaces modified with cRGDfK compared to surfaces modified with other peptide sequences tested. PAPA-coated polystyrene flasks with coupled cRGDfK (cRGDfK-PAPA) were then used for long-term studies of three hPSC lines (H9, hiPS-NHF1.3, Genea-02). Cell lines maintained for ten passages on cRGDfK-PAPA were assessed for colony morphology, proliferation rate, maintenance of OCT4 expression, cell viability at harvest, teratoma formation potential, and global gene expression as assessed by the PluriTest™ assay. cRGDfK-PAPA and control cultures maintained on Geltrex™ produced comparable results in most assays. No karyotypic abnormalities were detected in cultures maintained on cRGDfK-PAPA, while abnormalities were detected in cultures maintained on Geltrex™, StemAdhere™ or Synthemax™. This is the first report of long term maintenance of hPSC cultures on the scalable, stable, and cost-effective cRGDfK-PAPA coating.
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Técnicas de Cultura de Células/métodos , Materiais Revestidos Biocompatíveis , Peptídeos Cíclicos , Células-Tronco Pluripotentes/fisiologia , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Meios de Cultura/química , Perfilação da Expressão Gênica , Humanos , Inoculações SeriadasRESUMO
Characterisation of mouse pluripotent stem cells has revealed two distinct pluripotent states, naive and primed, that maintain characteristics of the pre and post implanted epiblast respectively. Recent studies have developed several culture systems that seek to recapitulate the naive phenomenon in human pluripotent stem cells. Therefore, robust methods to isolate these cells will be fundamental to assess their potential in modelling human development and disease. Here we review current methods for human naive pluripotent culture and collate a list of cell surface antigens that have been identified as markers to differentiate naive from primed human pluripotent stem cells. While these culture systems do display marker variability, and not all antigens mentioned were assessed in all methods, this review provides a resource for researchers of the human naive pluripotent stem cell state. SSEA-4, SSEA-3, CD24, CD75, CD7, CD77, CD130/GP130, CD57, CD90 and NLGN4X were all found to have a +/- expression profile in at least 2 methods, while +/- expression of Tra-1-81, CDH3, CD172a, CD107b, CD229 was reported in one method. Often it was reported that naive and primed cells could be defined using a low/medium/high expression of the following antigens TRA-1-60, PCDH1, GPR64, MHC Class I, however these markers were more likely to display expression pattern differences between methods. Studies using mouse naive cells indicate that they may have benefits over primed cells in modelling development and disease, and while it is yet to be determined if the same can be said about a human naive state, tools to identify this population should greatly further the field.
Assuntos
Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Fagocitose , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Humanos , Macrófagos/citologia , Células-Tronco Pluripotentes/citologiaRESUMO
AIMS: Expression of the HER2 oncogene in breast cancer is associated with resistance to treatment, and Her2 may regulate bioenergetics. Therefore, we investigated whether disruption of the electron transport chain (ETC) is a viable strategy to eliminate Her2high disease. RESULTS: We demonstrate that Her2high cells and tumors have increased assembly of respiratory supercomplexes (SCs) and increased complex I-driven respiration in vitro and in vivo. They are also highly sensitive to MitoTam, a novel mitochondrial-targeted derivative of tamoxifen. Unlike tamoxifen, MitoTam efficiently suppresses experimental Her2high tumors without systemic toxicity. Mechanistically, MitoTam inhibits complex I-driven respiration and disrupts respiratory SCs in Her2high background in vitro and in vivo, leading to elevated reactive oxygen species production and cell death. Intriguingly, higher sensitivity of Her2high cells to MitoTam is dependent on the mitochondrial fraction of Her2. INNOVATION: Oncogenes such as HER2 can restructure ETC, creating a previously unrecognized therapeutic vulnerability exploitable by SC-disrupting agents such as MitoTam. CONCLUSION: We propose that the ETC is a suitable therapeutic target in Her2high disease. Antioxid. Redox Signal. 26, 84-103.
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Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos/química , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Molecular , Terapia de Alvo Molecular , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Tamoxifeno/farmacologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0119549.].
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Autophagy favors both cell survival and cancer suppression, and increasing evidence reveals that microRNAs (MIRs) regulate autophagy. Previously we reported that MIR126 is downregulated in malignant mesothelioma (MM). Therefore, we investigated the role of MIR126 in the regulation of cell metabolism and autophagy in MM models. We report that MIR126 induces autophagic flux in MM cells by downregulating insulin receptor substrate-1 (IRS1) and disrupting the IRS1 signaling pathway. This was specific to MM cells, and was not observed in non-malignant cells of mesothelial origin or in MM cells expressing MIR126-insensitive IRS1 transcript. The MIR126 effect on autophagy in MM cells was recapitulated by IRS1 silencing, and antagonized by IRS1 overexpression or antisense MIR126 treatment. The MIR126-induced loss of IRS1 suppressed glucose uptake, leading to energy deprivation and AMPK-dependent phosphorylation of ULK1. In addition, MIR126 stimulated lipid droplet accumulation in a hypoxia-inducible factor-1α (HIF1α)-dependent manner. MIR126 also reduced pyruvate dehydrogenase kinase (PDK) and acetyl-CoA-citrate lyase (ACL) expression, leading to the accumulation of cytosolic citrate and paradoxical inhibition of pyruvate dehydrogenase (PDH) activity. Simultaneous pharmacological and genetic intervention with PDK and ACL activity phenocopied the effects of MIR126. This suggests that in MM MIR126 initiates a metabolic program leading to high autophagic flux and HIF1α stabilization, incompatible with tumor progression of MM. Consistently, MIR126-expressing MM cells injected into immunocompromised mice failed to progress beyond the initial stage of tumor formation, showing that increased autophagy has a protective role in MM.
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Autofagia/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Substratos do Receptor de Insulina/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/metabolismo , Mesotelioma/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genética , Transplante HeterólogoRESUMO
The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27(Kip1) (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157--a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27(KIP1) potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc.
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Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Interferon gama/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Senescência Celular/fisiologia , Chlorocebus aethiops , Quinase 2 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Fosforilação , Ligação ProteicaRESUMO
Malignant mesothelioma (MM) is an aggressive type of tumour causing high mortality. One reason for this paradigm may be the existence of a subpopulation of tumour-initiating cells (TICs) that endow MM with drug resistance and recurrence. The objective of this study was to identify and characterise a TIC subpopulation in MM cells, using spheroid cultures, mesospheres, as a model of MM TICs. Mesospheres, typified by the stemness markers CD24, ABCG2 and OCT4, initiated tumours in immunodeficient mice more efficiently than adherent cells. CD24 knock-down cells lost the sphere-forming capacity and featured lower tumorigenicity. Upon serial transplantation, mesospheres were gradually more efficiently tumrigenic with increased level of stem cell markers. We also show that mesospheres feature mitochondrial and metabolic properties similar to those of normal and cancer stem cells. Finally, we show that mesothelioma-initiating cells are highly susceptible to mitochondrially targeted vitamin E succinate. This study documents that mesospheres can be used as a plausible model of mesothelioma-initiating cells and that they can be utilised in the search for efficient agents against MM.
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Antineoplásicos/farmacologia , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores Tumorais/metabolismo , Antígeno CD24/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Técnicas de Silenciamento de Genes , Humanos , Concentração Inibidora 50 , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Mesotelioma Maligno , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Tocoferóis/farmacologiaRESUMO
We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.
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Mitocôndrias/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citrato (si)-Sintase/metabolismo , DNA Mitocondrial/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transplante HomólogoRESUMO
AIMS: To assess the effect of mitochondrially targeted vitamin E (VE) analogs on mitochondrial function and biogenesis. RESULTS: Mitochondrially targeted vitamin E succinate (MitoVES) is an efficient inducer of apoptosis in cancer cells. Here, we show that unlike its untargeted counterpart α-tocopheryl succinate, MitoVES suppresses proliferation of cancer cells at sub-apoptotic doses by way of affecting the mitochondrial DNA (mtDNA) transcripts. We found that MitoVES strongly suppresses the level of the displacement loop transcript followed by those of mtDNA genes coding for subunits of mitochondrial complexes. This process is coupled to the inhibition of mitochondrial respiration, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. In addition, exposure of cancer cells to MitoVES led to decreased expression of TFAM and diminished mitochondrial biogenesis. The inhibition of mitochondrial transcription was replicated in vivo in a mouse model of HER2(high) breast cancer, where MitoVES lowered the level of mtDNA transcripts in cancer cells but not in normal tissue. INNOVATION: Our data show that mitochondrially targeted VE analogs represent a novel class of mitocans that not only induce apoptosis at higher concentrations but also block proliferation and suppress normal mitochondrial function and transcription at low, non-apoptogenic doses. CONCLUSIONS: Our data indicate a novel, selective anti-cancer activity of compounds that act by targeting mitochondria of cancer cells, inducing significant alterations in mitochondrial function associated with transcription of mtDNA-coded genes. These changes subsequently result in the arrest of cell proliferation.
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DNA Mitocondrial/metabolismo , Mitocôndrias/efeitos dos fármacos , Receptor ErbB-2/genética , alfa-Tocoferol/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mitocôndrias/fisiologia , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
In Parkinson's disease and some atypical Parkinson's syndromes, aggregation of the α-synuclein protein (α-syn) has been linked to neurodegeneration. Many triggers for pathological α-syn aggregation have been identified, including port-translational modifications, oxidative stress and raised metal ions, such as Ca2+. Recently, it has been found using cell culture models that transient increases of intracellular Ca2+ induce cytoplasmic α-syn aggregates. Ca2+-dependent α-syn aggregation could be blocked by the Ca2+ buffering agent, BAPTA-AM, or by the Ca2+ channel blocker, Trimethadione. Furthermore, a greater proportion of cells positive for aggregates occurred when both raised Ca2+ and oxidative stress were combined, indicating that Ca2+ and oxidative stress cooperatively promote α-syn aggregation. Current on-going work using a unilateral mouse lesion model of Parkinson's disease shows a greater proportion of calbindin-positive neurons survive the lesion, with intracellular α-syn aggregates almost exclusively occurring in calbindin-negative neurons. These and other recent findings are reviewed in the context of neurodegenerative pathologies and suggest an association between raised Ca2+, α-syn aggregation and neurotoxicity.
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Cálcio/metabolismo , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Humanos , Espaço Intracelular/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/terapia , Ligação Proteica , Multimerização Proteica , alfa-Sinucleína/químicaRESUMO
AIMS: MiR126 was found to be frequently lost in many types of cancer, including malignant mesothelioma (MM), which represents one of the most challenging neoplastic diseases. In this study, we investigated the potential tumor suppressor function of MiR126 in MM cells. The effect of MiR126 was examined in response to oxidative stress, aberrant mitochondrial function induced by inhibition of complex I, mitochondrial DNA (mtDNA) depletion, and hypoxia. RESULTS: MiR126 was up-regulated by oxidative stress in nonmalignant mesothelial (Met5A) and MM (H28) cell lines. In Met5A cells, rotenone inhibited MiR126 expression, but mtDNA depletion and hypoxia up-regulated MiR126. However, these various stimuli suppressed the levels of MiR126 in H28 cells. MiR126 affected mitochondrial energy metabolism, reduced mitochondrial respiration, and promoted glycolysis in H28 cells. This metabolic shift, associated with insulin receptor substrate-1 (IRS1)-modulated ATP-citrate lyase deregulation, resulted in higher ATP and citrate production. These changes were linked to the down-regulation of IRS1 by ectopic MiR126, reducing Akt signaling and inhibiting cytosolic sequestration of Forkhead box O1 (FoxO1), which promoted the expression of genes involved in gluconeogenesis and oxidative stress defense. These metabolic changes induced hypoxia-inducible factor-1α (HIF1α) stabilization. Consequently, MiR126 suppressed the malignancy of MM cells in vitro, a notion corroborated by the failure of H28(MiR126) cells to form tumors in nude mice. INNOVATION AND CONCLUSION: MiR126 affects mitochondrial energy metabolism, resulting in MM tumor suppression. Since MM is a fatal neoplastic disease with a few therapeutic options, this finding is of potential translational importance.
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Proteínas Substratos do Receptor de Insulina/genética , Neoplasias Pulmonares/genética , Mesotelioma/genética , MicroRNAs/genética , Mitocôndrias/metabolismo , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos Nus , Transplante de Neoplasias , Estresse Oxidativo , Consumo de Oxigênio , Interferência de RNA , Transdução de SinaisRESUMO
Tumor-initiating cells (TICs) often survive therapy and give rise to second-line tumors. We tested the plausibility of sphere cultures as models of TICs. Microarray data and microRNA data analysis confirmed the validity of spheres as models of TICs for breast and prostate cancer as well as mesothelioma cell lines. Microarray data analysis revealed the Trp pathway as the only pathway upregulated significantly in all types of studied TICs, with increased levels of indoleamine-2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme of Trp metabolism along the kynurenine pathway. All types of TICs also expressed higher levels of the Trp uptake system consisting of CD98 and LAT1 with functional consequences. IDO1 expression was regulated via both transcriptional and posttranscriptional mechanisms, depending on the cancer type. Serial transplantation of TICs in mice resulted in gradually increased IDO1. Mitocans, represented by α-tocopheryl succinate and mitochondrially targeted vitamin E succinate (MitoVES), suppressed IDO1 in TICs. MitoVES suppressed IDO1 in TICs with functional mitochondrial complex II, involving transcriptional and posttranscriptional mechanisms. IDO1 increase and its suppression by VE analogues were replicated in TICs from primary human glioblastomas. Our work indicates that IDO1 is increased in TICs and that mitocans suppress the protein.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Mitocôndrias/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , alfa-Tocoferol/farmacologia , Linhagem Celular Tumoral , Complexo II de Transporte de Elétrons/genética , Complexo II de Transporte de Elétrons/metabolismo , Feminino , Proteína-1 Reguladora de Fusão/genética , Proteína-1 Reguladora de Fusão/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de Sinais , Triptofano/metabolismoRESUMO
UNLABELLED: Malignant mesothelioma (MM) is a fatal neoplastic disease with no therapeutic option. Therefore, the search for novel therapies is of paramount importance. METHODS: Since mitochondrial targeting of α-tocopheryl succinate (α-TOS) by its tagging with triphenylphosphonium enhances its cytotoxic effects to cancer cells, we tested its effect on MM cells and experimental mesotheliomas. RESULTS: Mitochondrially targeted vitamin E succinate (MitoVES) was more efficient in killing MM cells than α-TOS with IC50 lower by up to two orders of magnitude. Mitochondrial association of MitoVES in MM cells was documented using its fluorescently tagged analogue. MitoVES caused apoptosis in MM cells by mitochondrial destabilization, resulting in the loss of mitochondrial membrane potential, generation of reactive oxygen species, and destabilization of respiratory supercomplexes. The role of the mitochondrial complex II in the activity of MitoVES was confirmed by the finding that MM cells with suppressed succinate quinone reductase were resistant to MitoVES. MitoVES suppressed mesothelioma growth in nude mice with high efficacy. DISCUSSION: MitoVES is more efficient in killing MM cells and suppressing experimental mesotheliomas compared with the non-targeted α-TOS, giving it a potential clinical benefit.
Assuntos
Antineoplásicos/uso terapêutico , Mesotelioma/tratamento farmacológico , alfa-Tocoferol/análogos & derivados , alfa-Tocoferol/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/genética , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Estrutura Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , alfa-Tocoferol/farmacocinéticaRESUMO
α-Tocopheryl succinate (α-TOS) is a promising anti-cancer agent due to its selectivity for cancer cells. It is important to understand whether long-term exposure of tumour cells to the agent will render them resistant to the treatment. Exposure of the non-small cell lung carcinoma H1299 cells to escalating doses of α-TOS made them resistant to the agent due to the upregulation of the ABCA1 protein, which caused its efflux. Full susceptibility of the cells to α-TOS was restored by knocking down the ABCA1 protein. Similar resistance including ABCA1 gene upregulation was observed in the A549 lung cancer cells exposed to α-TOS. The resistance of the cells to α-TOS was overcome by its mitochondrially targeted analogue, MitoVES, that is taken up on the basis of the membrane potential, bypassing the enhanced expression of the ABCA1 protein. The in vitro results were replicated in mouse models of tumours derived from parental and resistant H1299 cells. We conclude that long-term exposure of cancer cells to α-TOS causes their resistance to the drug, which can be overcome by its mitochondrially targeted counterpart. This finding should be taken into consideration when planning clinical trials with vitamin E analogues.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , alfa-Tocoferol/uso terapêutico , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , CamundongosRESUMO
Oligodendroglial inclusion bodies characterize a subset of neurodegenerative diseases. Multiple system atrophy (MSA) is characterized by α-synuclein glial cytoplasmic inclusions and progressive supranuclear palsy (PSP) is associated with glial tau inclusions. The ubiquitin homologue, SUMO-1, has been identified in inclusion bodies in MSA, located in discrete sub-domains in α-synuclein-positive inclusions. We investigated SUMO-1 associated with oligodendroglial inclusion bodies in brain tissue from MSA and PSP and in glial cell models. We examined MSA and PSP cases and compared to age-matched normal controls. Fluorescence immunohistochemistry revealed frequent SUMO-1 sub-domains within and surrounding inclusions bodies in both diseases and showed punctate co-localization of SUMO-1 and the lysosomal marker, cathepsin D, in affected brain regions. Cell counting data revealed that 70-75 % of lysosomes in inclusion body-positive oligodendrocytes were SUMO-1-positive consistently across MSA and PSP cases, compared to 20 % in neighbouring inclusion body negative oligodendrocytes and 10 % in normal brain tissue. Hsp90 co-localized with some SUMO-1 puncta. We examined the SUMO-1 status of lysosomes in 1321N1 human glioma cells over-expressing α-synuclein and in immortalized rat oligodendrocyte cells over-expressing the four repeat form of tau following treatment with the proteasome inhibitor, MG132. We also transfected 1321N1 cells with the inherently aggregation-prone huntingtin exon 1 mutant, HttQ74-GFP. Each cell model showed the association of SUMO-1-positive lysosomes around focal cytoplasmic accumulations of α-synuclein, tau or HttQ74-GFP, respectively. Association of SUMO-1 with lysosomes was also detected in glial cells bearing α-synuclein aggregates in a rotenone-lesioned rat model. SUMO-1 labelling of lysosomes showed a major increase between 24 and 48 h post-incubation of 1321N1 cells with MG132 resulting in an increase in a 90 kDa SUMO-1-positive band that was immunopositive for Hsp90 and immunoprecipitated with an anti-SUMO-1 antibody. That SUMO-1 co-localizes with a subset of lysosomes in neurodegenerative diseases with glial protein aggregates and in glial cell culture models of protein aggregation suggests a role for SUMO-1 in lysosome function.