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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(1): 275-282, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32027289

RESUMO

OBJECTIVE: To investigate the effect of prostaglandin E2 recoptor 4 antagonist (EP4A) on the self-renewal ability of human CD34+ cells and its mechamism. METHODS: The peripheral blood hematopoietic stem cell of 20 healthy donors received the G-CSF-mobilization were collected, then the human CD34+ cells were sorted out by MACS microbead kit. The human CD34+ cells were treated with DMSO (control group), EP4A (EP4A group) and EP4A+EP4A antagonist (EP4A+EP4A group) for 72 hours. The differential genes and pathways related with CD34+ cell stemness were detected by Thermogram and Pathway enrichment analysis. and then the expression levels of protein and gene (ß-catenin, Nanog, Oct4, Sox2, Stat3, AKT, P38) were detected by qRT-PCR and Western blot respectively. RESULTS: EP4A could elevate the mRNA and protein expression of ß-catenin, Nanog, Oct4, Sox2, in comparison with control group, however, mRNA and protein expression of STAT3, AKT, P38 were not changed. When human CD34+ cell were cultured with EP4A+XAV939 it was found that the mRNA and protein expression of ß-catenin was downregulated, moreover the mRNA and protein expression of Nanog, Oct4, Sox2 were reduced. CONCLUSION: EP4A can upregulate stemness factors-ß-catenin, Nanog, Oct4 and Sox2 in human CD34+ cell in vitro, but not STAT3, AKT and P38.


Assuntos
Receptores de Prostaglandina E Subtipo EP4/metabolismo , Antígenos CD34 , Movimento Celular , Fator Estimulador de Colônias de Granulócitos , Humanos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero , Prostaglandinas , RNA Mensageiro , Linfócitos T
2.
Gene ; 734: 144381, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31978510

RESUMO

Down-regulation of stemness genes expression is important in differentiation therapy against cancer stem cells (CSCs). The aim of this study was to evaluate the Oct4 , Sox2, Nanog, and C-myc expression in rat breast cancer stem cells (LA7) which treated with human ovarian follicular fluid (FF), replicative senescent fibroblast culture supernatant (P14), and 16 h serum starved fibroblast supernatant (16 h-SFS). The cells were exposed to these biological fluids for 24 h, 72 h, and 7 days. Stem-loop RT-qPCR assay was used to quantify the expression of above mentioned genes. Results showed that FF had the least cytotoxic effect on the LA7 cells. Except for Nanog gene, exposure of LA7 cell line to 16 h-SFS and P14 decreased significantly expression of the three other genes after 24 h (P < 0.05). Nanog and Sox2 genes expression was also decreased in LA7 cells which have been already treated with FF for 24 h. Moreover, compared to the control solution, the expression of Oct4 increased significantly after 7 days exposure to FF (P < 0.05). Annexin V-PE /7-AAD-, acridine orange/ethidium bromide staining and doubling time assays revealed apoptosis and necrosis induction by these biological fluids in LA7 cells. Moreover, in an in vitro model of metastasis assay, i.e., scratch test, these fluids exhibited anti-LA7 migration activity which culminated in 16 h-SFS treated cells. Generally, this study showed that FF, 16 h-SFS, and P14 have positive effects on down-regulation of Nanog, Oct4, Sox2 and C-myc expression, and consequently can increase the differentiation of breast cancer stem cells. For the first time, this study provided some evidence indicating that some biological fluids have potential to differentiate the CSCs, show anti- survival, growth-, and cell migration activity.


Assuntos
Líquidos Corporais/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/genética , Células-Tronco Neoplásicas , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Meios de Cultura/farmacologia , Regulação para Baixo , Feminino , Líquido Folicular/fisiologia , Genes myc , Humanos , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genética
3.
Nat Commun ; 11(1): 562, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992715

RESUMO

Cancer immunotherapy has emerged as a promising cancer treatment. However, the presence of immune-refractory tumor cells limits its clinical success by blocking amplification of anti-tumor immunity. Previously, we found that immune selection by immunotherapy drives the evolution of tumors toward multi-modal resistant and stem-like phenotypes via transcription induction of AKT co-activator TCL1A by NANOG. Here, we report a crucial role of HSP90A at the crossroads between NANOG-TCL1A axis and multi-aggressive properties of immune-edited tumor cells by identifying HSP90AA1 as a NANOG transcriptional target. Furthermore, we demonstrate that HSP90A potentiates AKT activation through TCL1A-stabilization, thereby contributing to the multi-aggressive properties in NANOGhigh tumor cells. Importantly, HSP90 inhibition sensitized immune-refractory tumor to adoptive T cell transfer as well as PD-1 blockade, and re-invigorated the immune cycle of tumor-reactive T cells. Our findings implicate that the HSP90A-TCL1A-AKT pathway ignited by NANOG is a central molecular axis and a potential target for immune-refractory tumor.


Assuntos
Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Imunidade , Imunoterapia , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Isoxazóis/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Homeobox Nanog/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Resorcinóis/farmacologia
4.
Mater Sci Eng C Mater Biol Appl ; 106: 110259, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753381

RESUMO

Polymeric hydrogel-based 3D scaffolds are well-known structures, being used for cultivation and differentiation of stem cells. However, scalable systems that provide a native-like microenvironment with suitable biological and physical properties are still needed. Incorporation of nanomaterials into the polymeric systems is expected to influence the physical properties of the structure but also the stem cells fate. Here, alginate/gelatin hydrogel beads incorporated with mesoporous silica nanoparticles (MSNs) (average diameter 80.9 ±â€¯10 nm) and various surface chemistries were prepared. Human adipose-derived mesenchymal stem cells (hASCs) were subsequently encapsulated into the alginate/gelatin/silica hydrogels. Incorporation of amine- and carboxyl-functionalized MSNs (A-MSNs and C-MSNs) significantly enhances the stability of the hydrogel beads. In addition, the expression levels of Nanog and OCT4 imply that the incorporation of A-MSNs into the alginate/gelatin beads significantly improves the proliferation and the stemness of encapsulated hASCs. Importantly, our findings show that the presence of A-MSNs slightly suppresses in vivo inflammation. In contrast, the results of marker gene expression analyses indicate that cultivation of hASCs in alginate beads incorporated with C-MSNs (10% w/w) leads to a heterogeneously differentiated population of the cells, i.e., osteocytes, chondrocytes, and adipocytes, which is not appropriate for both cell culture and differentiation applications.


Assuntos
Técnicas de Cultura de Células/métodos , Hidrogéis/química , Nanopartículas/química , Dióxido de Silício/química , Tecido Adiposo/citologia , Alginatos/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Gelatina/química , Humanos , Hidrogéis/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Porosidade , Ratos , Ratos Wistar , Tecidos Suporte/química
5.
Tissue Cell ; 60: 21-24, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31582014

RESUMO

Cancer stem cells (CSCs) have been implicated in growth, metastasis, recurrence and chemo-/radio-resistance in several cancer types. Despite a plenty of literature about different in vitro techniques to enrich/isolate CSCs, their comparative characterization for stemness is not well established. In the present study, cells obtained following three in vitro assays [clonogenic assay, tumorsphere assay (TSA) and single cell assay (SCA)] were compared for their cancer stem-like cell characteristics in human lung adenocarcinoma (A549) cells. Expression of the pluripotent (OCT4, NANOG) and lung cancer stem cell marker (CD166) genes were studied in these cells. Results showed that in comparison to cells obtained from routine culture (CC), the cells obtained from TSA showed significantly higher expression of OCT-4 and NANOG. These results were further validated with quantification of cell surface cancer stem cell markers i.e. CD44+/CD24- in the cells obtained from different methods, which were higher in TSA and SCA. Additionally, functional characterization of cancer stem-like cells (CSLCs) using ALDH assay showed the highest % of ALDH+ cells in TSA. These results were in agreement with higher resistance of these cells against 5-Fluorouracil suggesting higher fraction of CSLCs in TSA than the other assays. These results showed that TSA provides a better method to enrich CSLCs in A549 lung adenocarcinoma cells.


Assuntos
Técnicas In Vitro , Células-Tronco Neoplásicas/patologia , Células A549 , Biomarcadores Tumorais/metabolismo , Separação Celular , Humanos , Receptores de Hialuronatos/metabolismo , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo
6.
Nat Commun ; 10(1): 4444, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570708

RESUMO

Ectopic transcription factor expression enables reprogramming of somatic cells to pluripotency, albeit with generally low efficiency. Despite steady progress in the field, the exact molecular mechanisms that coordinate this remarkable transition still remain largely elusive. To better characterize the final steps of pluripotency induction, we optimized an experimental system where pluripotent stem cells are differentiated for set intervals before being reintroduced to pluripotency-supporting conditions. Using this approach, we identify a transient period of high-efficiency reprogramming where ectopic transcription factors, but not serum/LIF alone, rapidly revert cells to pluripotency with near 100% efficiency. After this period, cells reprogram with somatic-like kinetics and efficiencies. We identify a set of OCT4 bound cis-regulatory elements that are dynamically regulated during this transient phase and appear central to facilitating reprogramming. Interestingly, these regions remain hypomethylated during in vitro and in vivo differentiation, which may allow them to act as primary targets of ectopically induced factors during somatic cell reprogramming.


Assuntos
Diferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Fibroblastos , Regulação da Expressão Gênica , Genômica , Cinética , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco
7.
J Exp Clin Cancer Res ; 38(1): 416, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619256

RESUMO

BACKGROUND: At the beginning of tumorigenesis, newly born cancer cells must successfully avoid attack by the immune system. Although most abnormal cells are efficiently identified and destroyed by the immune system, particularly by NK cells, the molecular mechanisms by which newly born cancer cells evade NK cell surveillance are not fully understood. METHODS: NK cell resistance of highly tumorigenic population of human prostate cancer (PCa) cells were confirmed by xenograft in SCID mice with or without NK cell neutralization. The mechanisms by which the tumorigenic PCa cells evaded NK cell attack were investigated by RNAseq, ChIPseq, generation of several transformants and xenograft in SCID mice. RESULTS: Here, we show that PCa cells have a strengthened ability to escape NK cell attack due to NANOG, a pluripotent-related transcription factor, mediating the repression of ICAM1, a cell adhesion molecule, during tumorigenesis. Mechanistically, NANOG directly binds to the region upstream of ICAM1. As the binding between NANOG and the upstream ICAM1 region increases, p300 binding to this region is diminished, resulting in decreased ICAM1 expression. High NANOG expression confers PCa cells the ability to resist NK cell attack via the repression of ICAM1. Consistent with these results, low ICAM1 expression is significantly correlated with a high recurrence rate in patients with PCa. CONCLUSIONS: Our findings indicate that repression of ICAM1 is a critical mechanism by which cancer cells evade attack from NK cells during tumorigenesis. These results suggest a pivotal role of NANOG in establishing a gene expression profile for escaping the immune system.


Assuntos
Regulação Neoplásica da Expressão Gênica , Molécula 1 de Adesão Intercelular/metabolismo , Células Matadoras Naturais/imunologia , Proteína Homeobox Nanog/metabolismo , Neoplasias da Próstata/imunologia , Evasão Tumoral , Animais , Carcinogênese , Progressão da Doença , Humanos , Molécula 1 de Adesão Intercelular/genética , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Camundongos SCID , Proteína Homeobox Nanog/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas
8.
Nucleic Acids Res ; 47(19): 10115-10133, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31555818

RESUMO

Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.


Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição/genética , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Autorrenovação Celular/genética , Reprogramação Celular/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Mutação/genética , Proteína Homeobox Nanog/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Transfecção
9.
BMC Cancer ; 19(1): 864, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470802

RESUMO

BACKGROUND: Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior. METHODS: Bronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids. RESULTS: Data demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice. CONCLUSIONS: Human bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids.


Assuntos
Acetazolamida/administração & dosagem , Anticarcinógenos/administração & dosagem , Neoplasias Brônquicas/tratamento farmacológico , Tumor Carcinoide/tratamento farmacológico , Isotiocianatos/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Acetazolamida/farmacologia , Animais , Anticarcinógenos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Brônquicas/genética , Neoplasias Brônquicas/metabolismo , Tumor Carcinoide/genética , Tumor Carcinoide/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isotiocianatos/farmacologia , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Anim Sci J ; 90(11): 1417-1425, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31486245

RESUMO

Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti-inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging-related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 µM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE-treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency-related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE-treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.


Assuntos
Senescência Celular , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Quempferóis/farmacologia , Oócitos/fisiologia , Suínos/embriologia , Animais , Blastocisto/metabolismo , Técnicas de Cultura Embrionária , Feminino , Integrinas/genética , Integrinas/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
11.
Tumour Biol ; 41(8): 1010428319869101, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31423948

RESUMO

Stemness phenotype mammospheres established from cell lines and tissues taken from autopsy can be used to test and to identify the most sensitive drugs for chemotherapy. Therefore, the aim of the present study was isolation and characterization of cancer stem cells derived from MCF7, MDA-MB231, and SKBR3 breast cancer cell lines to demonstrate the stemness phenotypes of mammospheres generated for further their applications in therapeutic approaches. In this study, two luminal subtypes of cell lines, MCF7 and SKBR3 and a basal subtype cell line, MDA-MB-231, were chosen. Mammosphere culturing was implemented for breast cancer stem cells isolation and mammosphere formation efficiency. At the next step, CD44+/CD24- cell ratio, Oct4 and Nanog mRNA levels, proliferation rate, migration rate of mammospheres, and drug resistance (in third passage) were evaluated. In addition, tumorigenicity of mammospheres in the chick embryo model was evaluated and compared through the chick chorioallantoic membrane assay. Among mammospheres formed in all three cell lines, MCF7 had the highest mammosphere formation efficiency. CD24 marker (a differentiation marker for the breast cancer cells) was significantly reduced in the mammospheres generated from MCF7 and SKBR3, during three passages. Also, Oct4 and Nanog transcript levels were significantly higher in all three types of mammospheres, as compared with their cell lines. Proliferation, migration rate, and drug resistance of mammospheres generated from all three cell lines were found to be significantly higher. Tumorigenicity of MCF7 mammospheres was confirmed through tumor size measurement. Also, tumorigenicity of MCF7 and SKBR3 mammospheres was confirmed through more migration from ectoderm to mesoderm and endoderm. We succeeded to establish the technology that can be extended to tissue in the future. We have demonstrated a number of mammospheres can be generated from cell lines. Also, cells with different molecular features showed different stemness phenotypes.


Assuntos
Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Animais , Antígeno CD24/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Embrião de Galinha , Membrana Corioalantoide/citologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Receptores de Hialuronatos/metabolismo , Células MCF-7 , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/genética , Células Tumorais Cultivadas
12.
Anim Sci J ; 90(9): 1127-1141, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31298467

RESUMO

The present study sought to examine whether trichostatin A (TSA)-assisted epigenetic transformation of porcine bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) affects the transcriptional activities of pluripotency-related genes (Oct4, Nanog, c-Myc, Sox2 and Rex1), multipotent stemness-related gene (Nestin) and anti-apoptotic/anti-senescence-related gene (Survivin). Epigenetically transformed or non-transformed BM-MSCs that had been transcriptionally profiled by qRT-PCR and had been analysed for different stages of apoptosis progression provided a source of nuclear donor cells for the in vitro production of cloned pig embryos. TSA-mediated epigenomic modulation has been found to enhance the multipotency extent, stemness and intracellular anti-ageing properties of porcine BM-MSCs. This has been confirmed by the relative abundances for Nanog, c-Myc Rex1, Sox2 and Survivin mRNAs in TSA-exposed BM-MSCs that turned out to be significantly higher than those of TSA-unexposed BM-MSCs. Additionally, TSA-assisted epigenomic modulation of BM-MSCs did not impact the caspase-8 activity, Bax protein expression and the incidence of TUNEL-positive cells. In conclusion, the considerably elevated quantitative profiles of Sox2, Rex1, c-Myc, Nanog and Survivin mRNA transcripts seem to trigger improved reprogrammability of TSA-treated BM-MSC nuclei in cloned pig embryos that thereby displayed remarkably increased blastocyst formation rates as compared to those noticed for embryos derived from TSA-untreated BM-MSCs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Clonagem de Organismos , Epigenômica , Produtos do Gene rex/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Fatores de Transcrição SOXB1/genética , Survivina/genética , Suínos , Proteína X Associada a bcl-2/genética
13.
BMC Dev Biol ; 19(1): 13, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31272387

RESUMO

BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.


Assuntos
Blastocisto/citologia , Embrião de Mamíferos/embriologia , Camadas Germinativas/embriologia , Proteína Homeobox Nanog/metabolismo , Fatores de Transcrição SOXF/metabolismo , Animais , Benzamidas/farmacologia , Bovinos , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Camadas Germinativas/citologia , Fator Inibidor de Leucemia/biossíntese , Proteína Homeobox Nanog/genética , Proteína Quinase C/biossíntese , Fatores de Transcrição SOXF/genética , Transdução de Sinais/fisiologia , Proteína Wnt1/biossíntese
14.
Anim Sci J ; 90(9): 1149-1160, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322312

RESUMO

Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC-like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram both mouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additional studies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC-driven bovine technologies can be realized.


Assuntos
Bovinos , Reprogramação Celular , Vetores Genéticos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/genética
15.
BMC Res Notes ; 12(1): 370, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262352

RESUMO

OBJECTIVE: Redox homeostasis maintenance is essential to bring about cellular functions. Particularly, embryonic stem cells (ESCs) have high fidelity mechanisms for DNA repair, high activity of different antioxidant enzymes and low levels of oxidative stress. Although the expression and activity of antioxidant enzymes are reduced throughout the differentiation, the knowledge about the transcriptional regulation of genes involved in defense against oxidative stress is yet restricted. Since glutathione is a central component of a complex system involved in preserving cellular redox status, we aimed to study whether the expression of the glutathione reductase (Gsr) gene, which encodes an essential enzyme for cellular redox homeostasis, is modulated by the transcription factors critical for self-renewal and pluripotency of ESCs. RESULTS: We found that Gsr gene is expressed in ESCs during the pluripotent state and it was upregulated when these cells were induced to differentiate, concomitantly with Nanog decreased expression. Moreover, we found an increase in Gsr mRNA levels when Nanog was downregulated by a specific shRNA targeting this transcription factor in ESCs. Our results suggest that Nanog represses Gsr gene expression in ESCs, evidencing a role of this crucial pluripotency transcription factor in preservation of redox homeostasis in stem cells.


Assuntos
Glutationa Redutase/genética , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica , Genes Reporter , Glutationa Redutase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/antagonistas & inibidores , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
16.
In Vitro Cell Dev Biol Anim ; 55(7): 473-481, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31214928

RESUMO

Leptin, a metabolic hormone, regulates the reproductive functions responding to both nutritional and body conditions. Embryonic stem cells play important roles in reproductive technology, but their derivation can be challenging. In this study, we evaluated the derivation rates of mouse embryonic stem cell (mESC) line from blastocysts developing in embryo culture media supplemented with different leptin concentrations. The results showed that addition of leptin into the embryo culture medium supported the in vitro development of mouse embryo. The mESC line derivation rates for media treated with 0, 10, 50, and 100 ng/ml of leptin were 61.24 % (54/88), 84.96 % (42/50), 81.79 % (61/76), and 85.78 % (56/67), respectively. In addition, leptin treatment of blastocysts upregulated the expression levels of the trophectoderm marker Cdx2, whereas inner cell mass markers Oct-4 and Nanog were not affected. mESC lines derived after leptin treatment demonstrated hallmarks of pluripotency, such as alkaline phosphatase activity, expression of, OCT4, NANOG, and SSEA1, as well as the ability to form embryoid bodies and well-differentiated teratomas. In conclusion, leptin has a positive effect on the derivation rate of mouse embryonic stem cell lines which may be, in part, due to its effects on the development of the trophectoderm cell lineage in the embryo.


Assuntos
Blastocisto/citologia , Proliferação de Células/efeitos dos fármacos , Leptina/farmacologia , Células-Tronco Embrionárias Murinas/citologia , Teratoma/metabolismo , Animais , Fator de Transcrição CDX2/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária , Corpos Embrioides/citologia , Antígenos CD15/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína Homeobox Nanog/biossíntese , Fator 3 de Transcrição de Octâmero/biossíntese , Teratoma/induzido quimicamente
17.
Biomed Environ Sci ; 32(4): 272-280, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31217063

RESUMO

OBJECTIVE: To explore the protective effect of NANOG against hydrogen peroxide (H2O2) -induced cell damage in the human hair follicle mesenchymal stem cells (hHF-MSCs). METHODS: NANOG was expressed from a lentiviral vector, pLVX-IRES-ZsGreen. NANOG hHF-MSCs and vector hHF-MSCs were treated with 400 µmol/L hydrogen peroxide (H2O2) for 2 h, the cell survival rate, cell morphology, ROS production, apoptosis and expression of AKT, ERK, and p21 were determined and compared. RESULTS: Our results showed that NANOG could activate AKT and upregulate the expression of p-AKT, but not p-ERK. When treated with 400 µmol/L H2O2, NANOG hHF-MSCs showed higher cell survival rate, lower ROS production and apoptosis, higher expression of p-AKT, higher ratio of p-AKT/AKT. CONCLUSION: Our results suggest that NANOG could protect hHF-MSCs against cell damage caused by H2O2 through activating AKT signaling pathway.


Assuntos
Folículo Piloso/citologia , Células-Tronco Mesenquimais/metabolismo , Proteína Homeobox Nanog/metabolismo , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Peróxido de Hidrogênio , Lentivirus , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína Homeobox Nanog/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
18.
Asian Pac J Cancer Prev ; 20(6): 1649-1654, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31244283

RESUMO

Objective: Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral neoplasms. Finding molecular markers for predicting prognosis is a high priority. The core transcription factors, OCT4, SOX2, and NANOG that regulate embryonic stem cell pluripotency have been implicated in progression of various malignancies. The predictive value of these markers and their role in the development of OSCC is still controversial. In this study, we therefore evaluated their expression in OSCCs and adjacent non-tumor tissue. Methods: A total of 60 frozen tumor and adjacent non-tumor tissue samples from 30 patients with OSCC were examined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Clinical and pathological data of patients including tumor stage, lymph node metastasis and tumor grade were also recorded. Results: Expression of SOX2 was significantly higher in adjacent non-tumor as compared to tumor tissue (P=0.04). No statistically significant differences were found for expression of OCT4 (P=0.50) and NANOG (P=0.68). Also, there was no significant association between expression of OCT4, SOX2, and NANOG and clinical or pathological data (P>0.05), although slightly higher values were noted in patients without lymph node metastasis. Conclusion: Based on the present data, decreased expression of SOX2 is correlated with carcinogenesis in the oral cavity and development of OSCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Prognóstico , Fatores de Transcrição SOXB1/genética
19.
Curr Med Sci ; 39(3): 403-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31209810

RESUMO

Accumulation of lactate in tumor has been linked to poor prognosis of oral squamous cell carcinoma (OSCC), but the underlying mechanism remained largely uncertain. Previous studies have suggested that presence of cancer stem cells (CSCs) closely correlated with cellular malignancy of OSCC. Here, using 3D organoid culture model, we investigated whether lactate promoted CSCs phenotype in primary OSCC cells. We generated organoids using fresh OSCC specimens and verified that organoids recapitulated histopathology and cellular heterogeneity of parental tumor. Organoids were then transfected with a Wnt reporter to visualize Wnt activity. The sphere forming assay demonstrated that high Wnt activity functionally designated CSCs population in OSCC cells. Further investigations indicated that lactate treatment promoted Wnt activity and increased the expression of CSCs (i.e. CD133+ cells) in organoids. Moreover, silencing monocarboxylate transporter 1 (MCT1), the prominent path for lactate uptake in human tumor with siRNA significantly impaired organoid forming capacity of OSCC cells. Together, our study demonstrated that lactate can promote CSCs phenotype of OSCC, and MCT1 may be a therapeutic target against OSCC growth.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Ácido Láctico/farmacologia , Transportadores de Ácidos Monocarboxílicos/genética , Neoplasias Bucais/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Simportadores/genética , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Idoso , Proteína Axina/genética , Proteína Axina/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Masculino , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Via de Sinalização Wnt/genética
20.
BMC Cancer ; 19(1): 518, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146720

RESUMO

BACKGROUND: Prostate cancer displays different morphologies which, in turn, affect patient outcome. This fact prompted questions about the lineage relationship between differentiated, more treatable prostate adenocarcinoma and poorly differentiated, less treatable non-adenocarcinoma including small cell carcinoma, and the molecular mechanism underlying prostate cancer differentiation. METHODS: Newly available non-adenocarcinoma/small cell carcinoma PDX LuCaP lines were analyzed for expression of stem cell transcription factors (scTF) LIN28A, NANOG, POU5F1, SOX2, which are responsible for reprogramming or de-differentiation. cDNA of these genes were cloned from small cell carcinoma LuCaP 145.1 into expression vectors to determine if they could function in reprogramming. RESULTS: Expression of scTF was detected in small cell carcinoma LuCaP 93, 145.1, 145.2, and non-adenocarcinoma LuCaP 173.1, 173.2A. Transfection of scTF from LuCaP 145.1 altered the gene expression of prostate non-small cell carcinoma cells, as well as fibroblasts. The resultant cells grew in stem-like colonies. Of note was a 10-fold lower expression of B2M in the transfected cells. Low B2M was also characteristic of LuCaP 145.1. Conversely, B2M was increased when stem cells were induced to differentiate. CONCLUSIONS: This work suggested a pathway in the emergence of non-adenocarcinoma/small cell carcinoma from adenocarcinoma through activation of scTF genes that produced cancer de-differentiation.


Assuntos
Adenocarcinoma/patologia , Carcinoma de Células Pequenas/patologia , Linhagem da Célula/genética , Neoplasias da Próstata/patologia , Adenocarcinoma/genética , Carcinoma de Células Pequenas/genética , Desdiferenciação Celular/genética , Linhagem Celular , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Neoplasias da Próstata/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOXB1/genética , Microglobulina beta-2/genética
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