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1.
Methods Mol Biol ; 2224: 183-193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606215

RESUMO

The availability of mouse models that allow inducible long-term hematopoietic stem cell (LT-HSC)-specific gene deletion in adult mice has been limited. Therefore, analysis of gene function in adult LT-HSCs has mostly relied on models such as the interferon inducible Mx1-Cre model. Unfortunately, the Mx1-Cre strain has significant drawbacks due to lack of specificity towards the hematopoietic system, adverse effects of interferon induction on the interpretation of data, and Cre expression leakage. In this chapter, we will describe the use of other inducible models, the tamoxifen-inducible Cre-ERT and Cre-ERT2 strains, and how these mice can be used to study gene function in LT-HSC.


Assuntos
Células-Tronco Adultas/fisiologia , Inativação Gênica/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Deleção de Genes , Inativação Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Tamoxifeno/farmacologia
2.
J Leukoc Biol ; 109(5): 865-875, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33615540

RESUMO

Although type I IFNs (IFN-I) are important for the innate and adaptive immune responses to suppress viral replication, prolonged IFN-I signaling in macrophages suppresses the immune response. Nuclear receptor estrogen-related receptor γ (ERRγ) regulates the transcription of genes involved in endocrine and metabolic functions. However, the role of ERRγ in macrophage immune responses to viruses remains largely unknown. ERRγ expression was significantly induced in mouse bone marrow-derived macrophages (BMDMs) treated with polyinosinic-polycytidylic acid (poly(I:C)). Our results indicated that the induction of ERRγ expression by poly(I:C) is mediated through activation of the cytoplasmic dsRNA receptors, retinoic acid-inducible gene I and melanoma differentiation-associated protein 5. In BMDMs, overexpression of ERRγ significantly increased gene expression and secretion of the IFN-I genes, IFN-α and IFN-ß, whereas abolition of ERRγ significantly attenuated poly(I:C)-mediated IFN-I secretion. Chromatin immunoprecipitation assays and mutation analyses of the IFN-I promoters revealed that ERRγ regulates the transcription of IFN-α and IFN-ß by binding to a conserved ERR response element in each promoter region. Finally, GSK5182 significantly suppressed poly(I:C)-mediated induction of IFN-I gene expression and secretion in BMDMs. Taken together, these findings reveal a previously unrecognized role for ERRγ in the transcriptional control of innate and adaptive immune response to dsRNA virus replication.


Assuntos
Regulação da Expressão Gênica , Interferon Tipo I/genética , Macrófagos/metabolismo , Poli I-C/farmacologia , Receptores Estrogênicos/metabolismo , Animais , Proteína DEAD-box 58/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Receptores Estrogênicos/genética , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Fator de Transcrição AP-1/metabolismo
3.
Nat Genet ; 53(2): 230-242, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33526923

RESUMO

Noncoding RNAs are exquisitely titrated by the cellular RNA surveillance machinery for regulating diverse biological processes. The RNA exosome, the predominant 3' RNA exoribonuclease in mammalian cells, is composed of nine core and two catalytic subunits. Here, we developed a mouse model with a conditional allele to study the RNA exosome catalytic subunit DIS3. In DIS3-deficient B cells, integrity of the immunoglobulin heavy chain (Igh) locus in its topologically associating domain is affected, with accumulation of DNA-associated RNAs flanking CTCF-binding elements, decreased CTCF binding to CTCF-binding elements and disorganized cohesin localization. DIS3-deficient B cells also accumulate activation-induced cytidine deaminase-mediated asymmetric nicks, altering somatic hypermutation patterns and increasing microhomology-mediated end-joining DNA repair. Altered mutation patterns and Igh architectural defects in DIS3-deficient B cells lead to decreased class-switch recombination but increased chromosomal translocations. Our observations of DIS3-mediated architectural regulation at the Igh locus are reflected genome wide, thus providing evidence that noncoding RNA processing is an important mechanism for controlling genome organization.


Assuntos
Linfócitos B/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/genética , RNA não Traduzido/genética , Hipermutação Somática de Imunoglobulina/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/genética , Proteínas de Fluorescência Verde/genética , Camundongos Knockout , Camundongos Transgênicos , Mutação , Processamento Pós-Transcricional do RNA , Recombinação Genética , Tamoxifeno/farmacologia
4.
Cell Transplant ; 30: 963689721991477, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33522308

RESUMO

TRANSLATIONAL RELEVANCE: No prophylactic treatments for COVID-19 have been clearly proven and found. In this pandemic context, cancer patients constitute a particularly fragile population that would benefit the best from such treatments, a present unmet need. TMPRSS2 is essential for COVID-19 replication cycle and it is under androgen control. Estrogen and androgen receptor dependent cues converge on TMPRSS2 regulation through different mechanisms of action that can be blocked by the use of hormonal therapies. We believe that there is enough body of evidence to foresee a prophylactic use of hormonal therapies against COVID-19 and this hypothesis can be easily tested on cohorts of breast and prostate cancer patients who follow those regimens. In case of pandemic, if the protective effect of hormonal therapies will be proven on cancer patients, the use of specific hormonal therapies could be extended to other oncological groups and to healthy individuals to decrease the overall risk of infection by SARS-CoV-2.Given the COVID-19 coronavirus emergency, a special focus is needed on the impact of this rapidly spreading viral infection on cancer patients. Androgen receptor (AR) signaling in the transmembrane protease serine 2 (TMPRSS2) regulation is emerging as an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility. In our study, we analyzed AR and TMPRSS2 expression in 17,352 normal and 9,556 cancer tissues from public repositories and stratified data according to sex and age. The emerging picture is that some patient groups may be particularly susceptible to SARS-CoV-2 infection and may benefit from antiandrogen- or tamoxifen-based therapies. These findings are relevant to choose proper treatments in order to protect cancer patients from concomitant SARS-CoV-2 contagion and related symptoms and put forward the idea that hormonal therapies could be used as prophylactic agents against COVID-19.


Assuntos
Antagonistas de Receptores de Andrógenos/uso terapêutico , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/complicações , Antagonistas de Estrogênios/uso terapêutico , Neoplasias da Próstata/complicações , Tamoxifeno/uso terapêutico , Antagonistas de Receptores de Andrógenos/farmacologia , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , /metabolismo , Descoberta de Drogas , Antagonistas de Estrogênios/farmacologia , Feminino , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/análise , Receptores Androgênicos/metabolismo , Serina Endopeptidases/análise , Serina Endopeptidases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia
5.
Molecules ; 26(1)2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33406699

RESUMO

Conventional chemotherapies used for breast cancer (BC) treatment are non-selective, attacking both healthy and cancerous cells. Therefore, new technologies that enhance drug efficacy and ameliorate the off-target toxic effects exhibited by currently used anticancer drugs are urgently needed. Here we report the design and synthesis of novel mesoporous silica nanoparticles (MSNs) equipped with the hormonal drug tamoxifen (TAM) to facilitate guidance towards estrogen receptors (ERs) which are upregulated in breast tumours. TAM is linked to the MSNs using a poly-ʟ-histidine (PLH) polymer as a pH-sensitive gatekeeper, to ensure efficient delivery of encapsulated materials within the pores. XRD, HR-TEM, DLS, SEM, FT-IR and BET techniques were used to confirm the successful fabrication of MSNs. The MSNs have a high surface area (>1000 m2/g); and a mean particle size of 150 nm, which is an appropriate size to allow the penetration of premature blood vessels surrounding breast tumours. Successful surface functionalization was supported by FT-IR, XPS and TGA techniques, with a grafting ratio of approximately 29%. The outcomes of this preliminary work could be used as practical building blocks towards future formulations.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Dióxido de Silício/química , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/química , Composição de Medicamentos , Desenho de Fármacos , Descoberta de Drogas , Liberação Controlada de Fármacos , Feminino , Humanos , Nanopartículas/química , Porosidade , Tamoxifeno/química
6.
Cancer Sci ; 112(4): 1603-1613, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33453094

RESUMO

Breast cancer is the leading cause of cancer death in women. Hormone-receptor-positive breast cancer (HR + BC) is the most common pathological type of breast cancer, of which the main treatment method is endocrine therapy. Unfortunately, primary or acquired drug resistance greatly limits its efficacy. In recent years, the newly launched CDK4/6 inhibitors could effectively reverse endocrine resistance in breast cancer. However, considering their expensive price and side effects, it is particularly important to find out effective biomarkers and screen sensitive patients. Here, we found through bioinformatics analysis that high mobility group box 1 (HMGB1) expression increased in endocrine-resistant HR + BC. In clinical specimens, the higher expression of HMGB1 was associated with shorter progression-free survival (PFS) for HR + BC patients with endocrine therapy after surgery. For endocrine-resistant breast cancer, compared with HMGB1-negative patients, HMGB1-positive patients who received CDK4/6 inhibitors treatment benefited more in PFS. Moreover, we demonstrated that HMGB1 promoted tamoxifen resistance by combining with the Toll-like receptor 4 (TLR4) and activating nuclear factor kappa B (NF-κB) pathway. CDK4/6 inhibitors could downregulate the expression of HMGB1 and suppress the TLR4-NF-κB pathway, and in turn reverse tamoxifen resistance. These results illuminated the critical role of HMGB1 in the process of tamoxifen resistance, explained the mechanism of CDK4/6 inhibitors reversing tamoxifen resistance, and suggested the feasibility of HMGB1 as a potential biomarker for screening sensitive patients receiving CDK4/6 inhibitors.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteína HMGB1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Intervalo Livre de Progressão , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Nature ; 590(7845): 326-331, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33505018

RESUMO

Resistance to insulin and insulin-like growth factor 1 (IGF1) in pancreatic ß-cells causes overt diabetes in mice; thus, therapies that sensitize ß-cells to insulin may protect patients with diabetes against ß-cell failure1-3. Here we identify an inhibitor of insulin receptor (INSR) and IGF1 receptor (IGF1R) signalling in mouse ß-cells, which we name the insulin inhibitory receptor (inceptor; encoded by the gene Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to INSR and IGF1R4, and a mannose 6-phosphate receptor domain that is also found in the IGF2 receptor (IGF2R)5. Knockout mice that lack inceptor (Iir-/-) exhibit signs of hyperinsulinaemia and hypoglycaemia, and die within a few hours of birth. Molecular and cellular analyses of embryonic and postnatal pancreases from Iir-/- mice showed an increase in the activation of INSR-IGF1R in Iir-/- pancreatic tissue, resulting in an increase in the proliferation and mass of ß-cells. Similarly, inducible ß-cell-specific Iir-/- knockout in adult mice and in ex vivo islets led to an increase in the activation of INSR-IGF1R and increased proliferation of ß-cells, resulting in improved glucose tolerance in vivo. Mechanistically, inceptor interacts with INSR-IGF1R to facilitate clathrin-mediated endocytosis for receptor desensitization. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of inceptor resulted in the retention of inceptor and INSR at the plasma membrane to sustain the activation of INSR-IGF1R in ß-cells. Together, our findings show that inceptor shields insulin-producing ß-cells from constitutive pathway activation, and identify inceptor as a potential molecular target for INSR-IGF1R sensitization and diabetes therapy.


Assuntos
Glicemia/metabolismo , Antagonistas da Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Animais , Glicemia/análise , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Tamanho Celular , Clatrina/metabolismo , Células Endócrinas/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Teste de Tolerância a Glucose , Complexo de Golgi/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Proteínas de Neoplasias/química , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia
8.
Expert Opin Drug Metab Toxicol ; 17(3): 307-321, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33320718

RESUMO

Introduction: Tamoxifen is still an important drug in hormone-dependent breast cancer therapy. Personalization of its clinical use beyond hormone receptor positivity could improve the substantial variability of the treatment response.Areas covered: The overview of the current evidence for the treatment personalization using therapeutic drug monitoring, or using genetic biomarkers including CYP2D6 is provided. Although many studies focused on the PK aspects or the impact of CYP2D6 variability the translation into clinical routine is not clearly defined due to the inconsistent clinical outcome data.Expert opinion: We believe that at least the main candidate factors, i.e. CYP2D6 polymorphism, CYP2D6 inhibition, endoxifen serum levels may become important predictors of clinical relevance for tamoxifen treatment personalization in the future. To achieve this aim, however, further research should take into consideration more precise characterization of the disease, epigenetic factors and also utilize an appropriately powered multifactorial approach instead of a single gene evaluating studies.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/farmacocinética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Citocromo P-450 CYP2D6/genética , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Polimorfismo Genético , Medicina de Precisão , Receptores Estrogênicos/metabolismo , Tamoxifeno/farmacocinética , Resultado do Tratamento
9.
Methods Mol Biol ; 2158: 323-336, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32857384

RESUMO

Genetic lineage tracing is accomplished using bi-transgenic mice, where one allele is altered to express Cre recombinase, and another allele encodes a Cre-dependent genetic reporter protein. Once Cre is activated (constitutive or in response to tamoxifen), the marker gene-expressing cells become indelibly labeled by the reporter protein. Therefore, daughter cells derived from labeled cells are permanently labeled even if the marker gene that drove Cre recombinase expression is no longer expressed in these cells. This system is commonly used to label putative progenitor cells and determine the fate of their progeny. Here, we describe the use of c-kit-based genetic lineage-tracing mouse line as an example and discuss caveats for performing these types of experiments.


Assuntos
Linhagem da Célula/genética , Rastreamento de Células/métodos , Células-Tronco/química , Células-Tronco/metabolismo , Animais , Expressão Gênica , Genes Reporter , Ligação Genética , Proteínas de Fluorescência Verde/genética , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Miócitos Cardíacos/química , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/genética , Células-Tronco/citologia , Tamoxifeno/farmacologia
10.
Mol Med Rep ; 23(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33179087

RESUMO

The H19 long non­coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen­resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF­7/TAMR cell epithelial­mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19­mediated effects. Reverse transcription­quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit­8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF­7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E­cadherin and markedly increased the expression levels of mesenchymal marker N­cadherin in MCF­7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose­dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19­induced alterations in N­cadherin and E­cadherin expression levels. Curcumin also prevented H19­induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF­7/TAMR cells, suggesting that curcumin may prevent H19­associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer.


Assuntos
Neoplasias da Mama/genética , Curcumina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , RNA Longo não Codificante/genética , Tamoxifeno/farmacologia , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7
11.
Nat Commun ; 11(1): 5513, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139730

RESUMO

Cyclin D1 is one of the most important oncoproteins that drives cancer cell proliferation and associates with tamoxifen resistance in breast cancer. Here, we identify a lncRNA, DILA1, which interacts with Cyclin D1 and is overexpressed in tamoxifen-resistant breast cancer cells. Mechanistically, DILA1 inhibits the phosphorylation of Cyclin D1 at Thr286 by directly interacting with Thr286 and blocking its subsequent degradation, leading to overexpressed Cyclin D1 protein in breast cancer. Knocking down DILA1 decreases Cyclin D1 protein expression, inhibits cancer cell growth and restores tamoxifen sensitivity both in vitro and in vivo. High expression of DILA1 is associated with overexpressed Cyclin D1 protein and poor prognosis in breast cancer patients who received tamoxifen treatment. This study shows the previously unappreciated importance of post-translational dysregulation of Cyclin D1 contributing to tamoxifen resistance in breast cancer. Moreover, it reveals the novel mechanism of DILA1 in regulating Cyclin D1 protein stability and suggests DILA1 is a specific therapeutic target to downregulate Cyclin D1 protein and reverse tamoxifen resistance in treating breast cancer.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ciclina D1/genética , RNA Longo não Codificante/metabolismo , Tamoxifeno/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Pessoa de Meia-Idade , Prognóstico , Processamento de Proteína Pós-Traducional/genética , Estabilidade Proteica , Proteólise , RNA Longo não Codificante/genética , Tamoxifeno/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
12.
Nat Commun ; 11(1): 4061, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792541

RESUMO

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.


Assuntos
Aderências Teciduais/metabolismo , Aderências Teciduais/patologia , Animais , Benzofenonas/farmacologia , Sistemas CRISPR-Cas , Células Cultivadas , Doxiciclina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência , Gastroenteropatias/metabolismo , Gastroenteropatias/patologia , Humanos , Imuno-Histoquímica , Isoxazóis/farmacologia , Lipossomos/metabolismo , Camundongos , Células NIH 3T3 , Parabiose , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia
13.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32611759

RESUMO

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.


Assuntos
Ebolavirus/efeitos dos fármacos , Proteína C1 de Niemann-Pick/antagonistas & inibidores , Receptores Virais/antagonistas & inibidores , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas Virais de Fusão/antagonistas & inibidores , Vírion/efeitos dos fármacos , Animais , Sítios de Ligação , Bioensaio , Chlorocebus aethiops , Clomifeno/química , Clomifeno/farmacologia , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Proteína C1 de Niemann-Pick/química , Proteína C1 de Niemann-Pick/genética , Proteína C1 de Niemann-Pick/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/farmacologia , Toremifeno/química , Toremifeno/farmacologia , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo
14.
Breast Cancer Res ; 22(1): 80, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727562

RESUMO

BACKGROUND: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. EXPERIMENTAL DESIGN: To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. RESULTS: Here, for the first time, we provide an ample evidence that a high level of the detoxifying enzyme SULT1A1 confers resistance to TMX therapy in both in vitro and ex vivo models and correlates with TMX resistance in metastatic samples in relapsed patients. Based on the data from different approaches, we identified three anticancer compounds, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis), aminoflavone (AF), and oncrasin-1 (ONC-1), whose tumor cell inhibition activity is dependent on SULT1A1. We discovered thioredoxin reductase 1 (TrxR1, encoded by TXNRD1) as a target of bio-activated RITA, AF, and ONC-1. SULT1A1 depletion prevented the inhibition of TrxR1, induction of oxidative stress, DNA damage signaling, and apoptosis triggered by the compounds. Notably, RITA efficiently suppressed TMX-unresponsive patient-derived breast cancer cells ex vivo. CONCLUSION: We have identified a mechanism of resistance to TMX via hyperactivated SULT1A1, which renders selective vulnerability to anticancer compounds RITA, AF, and ONC-1, and provide a rationale for a new combination therapy to overcome TMX resistance in breast cancer patients. Our novel findings may provide a strategy to circumvent TMX resistance and suggest that this approach could be developed further for the benefit of relapsed breast cancer patients.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/química , Antineoplásicos Hormonais/farmacologia , Apoptose , Arilsulfotransferase/genética , Arilsulfotransferase/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Tamoxifeno/química , Células Tumorais Cultivadas
15.
Anticancer Res ; 40(8): 4529-4535, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727783

RESUMO

BACKGROUND/AIM: Although ginseng seed oil (GSO) appears to have various roles in the body, its anti-cancer effect has not been investigated. Tamoxifen is widely used to treat estrogen receptor-positive (ER+) breast cancer but shows adverse effects with drug resistance. This study investigated the effect of GSO in ER+ breast cancer cell growth. MATERIALS AND METHODS: Cell viability assays, western blots and Annexin V staining were conducted to examine cell viability and apoptosis. The synergistic effect of tamoxifen in combination with GSO or oleic acid (OA) was determined. RESULTS: GSO and OA caused apoptosis of MCF-7 ER+ breast cancer cells and had synergistic effects with tamoxifen in inhibiting tamoxifen-resistant MCF-7 (MCF-7TAMR) ER+ breast cancer cell growth. CONCLUSION: GSO may block ER+ breast cancer recurrence in combination with tamoxifen.


Assuntos
Neoplasias da Mama/metabolismo , Ácido Oleico/farmacologia , Panax/química , Óleos Vegetais/farmacologia , Receptores Estrogênicos/metabolismo , Tamoxifeno/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Células MCF-7 , Óleos Vegetais/química , Sementes/química
16.
Am J Physiol Renal Physiol ; 319(3): F423-F435, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32657158

RESUMO

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.


Assuntos
Túbulos Renais Distais/enzimologia , Recombinases/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Antagonistas de Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Recombinases/genética , Simportadores de Cloreto de Sódio/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Tamoxifeno/farmacologia
17.
Am J Chin Med ; 48(5): 1221-1241, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32668964

RESUMO

Tamoxifen is one of the most common hormone therapy drug for estrogen receptor (ER)-positive breast cancer. Tumor cells with drug resistance often cause recurrence and metastasis in cancer patients. Luteolin is a natural compound found from various types of vegetables and exhibit anticancer activity in different cancers. This study demonstrated that luteolin inhibits the proliferation and induces apoptosis of tamoxifen-resistant ER-positive breast cancer cells. Luteolin also causes cell cycle arrest at the G2/M phase and decreases mitochondrial membrane potential. Besides, luteolin reduces the levels of activated PI3K/AKT/mTOR signaling pathway. The combination treatment of luteolin and PI3K, AKT, or mTOR inhibitors synergistically increases apoptosis in tamoxifen-resistant ER-positive breast cancer cells. Ras gene family (K-Ras, H-Ras, and N-Ras), an activator of PI3K, was transcriptionally repressed by luteolin via induction of tumor suppressor mixed-lineage leukemia 3 (MLL3) expression. MLL3 increases the level of monomethylation of Histone 3 Lysine 4 on the enhancer and promoter region of Ras genes, thus causes repression of Ras expressions. Our finding implies that luteolin was a promising natural agent against tamoxifen resistance of breast cancer.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a DNA/fisiologia , Expressão Gênica/efeitos dos fármacos , Luteolina/farmacologia , Antineoplásicos Fitogênicos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Metilação/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Tamoxifeno/farmacologia , Proteínas ras/genética , Proteínas ras/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(26): 15047-15054, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32532922

RESUMO

Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example is JAK2 By binding to two specific sequences in the promoter, ZIP suppresses JAK2 expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ER-positive breast cancer.


Assuntos
Neoplasias da Mama/enzimologia , Proteínas Quinases Associadas com Morte Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Tamoxifeno/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proteínas Quinases Associadas com Morte Celular/genética , Feminino , Humanos , Janus Quinase 2/genética , Camundongos , Camundongos SCID , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Fator de Transcrição STAT3/genética
19.
Gene ; 755: 144906, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32554048

RESUMO

The olive flounder Paralichthys olivaceus, an important marine fish, shows gender differences in growth. The mechanism on its gonadal differentiation direction affected with exogenous factors still needs to be clarified. The anti-Müllerian hormone (amh) gene is involved in fish testicular differentiation and maintenance. The aim of this study was to investigate the expression of the flounder amh in tissues and the gonads. The quantitative expression analysis results showed that it was highly expressed in the testis, especially in the testis at stages I - IV (P < 0.05). Also, amh was detected in Sertoli cells surrounding spermatogonia and peripheral seminiferous lobule of the testis with in situ hybridization (ISH) and immunohistochemistry (IHC). During the differentiation period, the amh expression in the testis of the tamoxifen treatment group (100 ppm) was higher than that in the ovary of the 17ß-estradiol (E2, 5 ppm) group, and the expression levels of amh during process of the male differentiation in the tamoxifen group were higher than those in the 17ɑ-methyltestosterone (MT, 5 ppm) group (P < 0.05). ISH results also exhibited that amh was expressed in the somatic cells that surrounded the germ cells of juvenile flounder similar to adult ones. Furthermore, the flounder gonads in the tamoxifen group maintained more germ cells and somatic cells than those in the MT group from 20 to 80 mm total length (TL). Especially, at 60 and 80 mm TL, the numbers of germ and somatic cells in the tamoxifen group were significantly higher than those in the MT group (P < 0.05). In summary, amh might initiate the process of testicular differentiation, and is involved in the early development and maintenance of testis.


Assuntos
Hormônio Antimülleriano/genética , Linguado/genética , Células de Sertoli/metabolismo , Animais , Hormônio Antimülleriano/metabolismo , Diferenciação Celular , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/metabolismo , Hibridização In Situ/métodos , Masculino , Metiltestosterona/farmacologia , Ovário/metabolismo , Diferenciação Sexual/genética , Espermatogônias/metabolismo , Tamoxifeno/farmacologia , Testículo/metabolismo
20.
Nat Commun ; 11(1): 2781, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493900

RESUMO

Mutations disrupting regulatory T (Treg) cell function can cause IPEX and IPEX-related disorders, but whether established disease can be reversed by correcting these mutations is unclear. Treg-specific deletion of the chromatin remodeling factor Brg1 impairs Treg cell activation and causes fatal autoimmunity in mice. Here, we show with a reversible knockout model that re-expression of Brg1, in conjunction with the severe endogenous proinflammatory environment, can convert defective Treg cells into powerful, super-activated Treg cells (SuperTreg cells) that can resolve advanced autoimmunity,  with  Brg1 re-expression in a minor fraction of Treg cells sufficient for the resolution in some cases. SuperTreg cells have enhanced trafficking and regulatory capabilities, but become deactivated as the inflammation subsides, thus avoiding excessive immune suppression. We propose a simple, robust yet safe gene-editing-based therapy for IPEX and IPEX-related disorders that exploits the defective Treg cells and the inflammatory environment pre-existing in the patients.


Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças do Sistema Imunitário/congênito , Linfócitos T Reguladores/imunologia , Alelos , Animais , Citocinas/metabolismo , DNA Helicases/deficiência , Diabetes Mellitus Tipo 1/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Doenças do Sistema Imunitário/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR3/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Linfócitos T Reguladores/efeitos dos fármacos , Tamoxifeno/farmacologia , Fatores de Transcrição/deficiência
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