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1.
Anticancer Res ; 39(11): 6097-6105, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704837

RESUMO

BACKGROUND/AIM: Colorectal cancer (CRC) is one of the most common in the world and its prevalence is rapidly increasing. Jagged-1-activated Notch signaling by apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) promotes CRC, and high expression of Jagged-1 is associated with poor prognosis. However, its clinical implication is unknown. The aim of this study was to investigate the clinical role of Jagged-1-activated Notch signaling by APEX1. MATERIALS AND METHODS: The 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the anti-cancer efficacy of 5-fluorouracil (5-FU), oxaliplatin, and irinotecan. Tissue from CRC patients was analyzed to assess the clinical specificity of Jagged-1 activated by APEX1. RESULTS: The half-maximal inhibitory concentration (IC50) in cells co-expressing APEX1 and Jagged-1 cells was higher than that in cells expressing only APEX1. These results indicated that the simultaneous expression of APEX1 and Jagged-1 might be associated with chemoresistance toward 5-FU, oxaliplatin, and irinotecan. Analysis of tissue from CRC patients revealed that high expression of Jagged-1 was associated with a statistically significantly low response to chemotherapy. CONCLUSION: Overexpression of Jagged-1 by APEX1 might serve as a predictor of response to chemotherapy and of poor prognosis, and moreover may be a therapeutic target for chemotherapy of advanced CRC.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína Jagged-1/metabolismo , Receptor Notch1/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Feminino , Fluoruracila/administração & dosagem , Seguimentos , Humanos , Irinotecano/administração & dosagem , Proteína Jagged-1/genética , Masculino , Pessoa de Meia-Idade , Oxaliplatina/administração & dosagem , Prognóstico , Receptor Notch1/genética , Estudos Retrospectivos , Taxa de Sobrevida , Células Tumorais Cultivadas
2.
J Cancer Res Clin Oncol ; 145(11): 2835-2843, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31506740

RESUMO

PURPOSE: Molecular mechanisms of response to hypomethylating agents in patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) still remain largely unknown. Therefore, the effects of 5-Azacytidine (Aza) on clonal architecture and DNA methylation were investigated in this study. METHODS: Using next-generation sequencing (NGS), 30 myeloid leukemia-associated genes were analyzed in 15 MDS/CMML patients with excellent response to Aza. Effects on methylation levels were analyzed by quantitative methylation analysis using pyrosequencing for the global methylation marker LINE-1 in patients and myeloid cell lines. Various myeloid cell lines and a healthy cohort were screened for methylation levels in 23 genes. Selected targets were verified on the MDS/CMML cohort. RESULTS: The study presented here showed a stable variant allele frequency and stable global methylation levels in responding patients. A significant demethylation of EZH2 and NOTCH1 was revealed in patients with Aza response. CONCLUSIONS: A response to Aza is not associated with eradication of malignant clones, but rather with a stabilization of the clonal architecture. We suggest changes in CpG methylation levels of EZH2 and NOTCH1 as potential targets of epigenetic response to Aza treatment which may also serve as useful biomarkers after clinical evaluation.


Assuntos
Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielomonocítica Crônica/genética , Síndromes Mielodisplásicas/genética , Receptor Notch1/genética , Idoso , Antimetabólitos Antineoplásicos/farmacologia , Estudos de Casos e Controles , Ilhas de CpG , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Leucemia Mielomonocítica Crônica/patologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia , Prognóstico , Receptor Notch1/metabolismo , Células Tumorais Cultivadas
3.
Gene ; 715: 144029, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31376409

RESUMO

Intervertebral disc degeneration (IDD) is a major cause of lower back pain, but the specific molecular mechanisms governing its development are poorly characterized. This study sought to assess to what extent HOTAIR, a long non-coding (Lnc) RNA is expressed in IDD and regulates the apoptotic death of nucleus pulposus (NP) cells. We therefore used real-time qPCR to measure HOTAIR and microRNA(miR)-34a-5p in degenerative NP cells, and then validated their functional relevance via overexpressing them in these NP cells. We further verified the targets of these RNA constructs in 293 T cells through the use of a dual luciferase reporter assay. We further measured NP cell apoptosis via flow cytometry and Notch1 expression via western blotting. Our results indicated that IDD was linked with decreased HOTAIR expression relative to regular NP cells, and overexpressing this lncRNA was linked to reduced apoptotic NP cell death, whereas overexpressing miR-34a-5p had the opposite effect. We found that HOTAIR served as a miR-34a-5p sponge, sequestering this miRNA and thereby down regulating genes linked to apoptosis through the Notch signaling pathway. Even in naturally degenerated NP cells, HOTAIR delayed the onset of apoptosis. Together these results reveal that a HOTAIR/miR-34a-5p/Notch1 signaling pathway may regulate the development of IDD, potentially making HOTAIR a viable target for treatment of this disease.


Assuntos
Apoptose , Regulação para Baixo , Degeneração do Disco Intervertebral/metabolismo , MicroRNAs/biossíntese , RNA Longo não Codificante/biossíntese , Receptor Notch1/biossíntese , Transdução de Sinais , Adulto , Idoso , Feminino , Humanos , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Núcleo Pulposo/patologia , RNA Longo não Codificante/genética , Receptor Notch1/genética
4.
Anticancer Res ; 39(8): 4165-4170, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366501

RESUMO

AIM: To examine the influence of hypoxia on the in vitro growth of leukaemia cells and the activity of signalling proteins to better understand the pathophysiology of leukaemia cells in human bone marrow. MATERIALS AND METHODS: Six human leukaemia cell lines were cultured under normoxic or hypoxic conditions. Cell growth, recovery of clonogenic cells, and the expression and activation of various signalling proteins were examined. RESULTS: Hypoxia suppressed cell growth and the recovery of clonogenic cells. Moreover, hypoxia up-regulated hypoxia-inducible factor (HIF) 1α and HIF2α expression while suppressing the expression and activation of NOTCH1, mechanistic target of rapamycin kinase (mTOR) activation, and nuclear factor-kappa B (NF-κB) phosphorylation. CONCLUSION: We found that hypoxia up-regulated HIF expression while it suppressed the self-renewal capacity of leukaemia cells, NOTCH activity, and expression of its down-stream signalling molecules, which differs from previous reports mentioning that HIF activates NOTCH signalling. Our findings serve to further elucidate the in vivo pathophysiology of leukaemia cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leucemia/genética , Receptor Notch1/genética , Ciclo Celular/genética , Hipóxia Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia/patologia , NF-kappa B/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
5.
Gene ; 715: 143970, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31330235

RESUMO

BACKGROUND: Bicuspid aortic valve (BAV) formation is genetically determined, with reduced penetrance and variable expressivity. NOTCH1 is a proven candidate gene and its mutations have been found in familial and sporadic cases of BAV. METHODS: 66 BAV patients from the GISSI VAR study were genotyped for the NOTCH1 gene. RESULTS: We identified 63 variants, in heterozygous and homozygous states. Fifty-two are common polymorphisms present in almost all patients. Eleven variants are new and never yet reported: two are non-synonymous substitutions, Gly540Asp in exon 10 and Glu851Gln in exon 16; one is in the 3'UTR region and seven in introns, one corresponds to a T allele insertion in intron 27. We selected four statistically noteworthy and seven new variants identified in six BAV patients and correlated them with clinical and demographic variables and with imaging and histological parameters. Preliminary data show that four were BAV patients with isolated stenosis in patients over 60 aged. These variants may correlate with a later need for surgery for the presence of stenosis and not aortic valve regurgitation or ascending aortic aneurysm. CONCLUSIONS: Completing the genotyping of 62 BAV patients we found 11 new variants in the NOTCH1 gene never yet reported. These findings confirm that the identification of new, clinically remarkable biomarkers for BAV requires a deeper genetic understanding of the NOTCH1 gene variants, which could be targeted by future diagnostic and therapeutic strategies.


Assuntos
Estenose da Valva Aórtica/genética , Valva Aórtica/anormalidades , Doenças das Valvas Cardíacas , Mutação de Sentido Incorreto , Penetrância , Receptor Notch1/genética , Adulto , Alelos , Substituição de Aminoácidos , Éxons , Feminino , Heterozigoto , Homozigoto , Humanos , Íntrons , Itália , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Análise de Sequência de DNA
6.
Fish Shellfish Immunol ; 92: 680-689, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31271837

RESUMO

The Notch signaling pathway is known to regulate innate immunity by influencing macrophage function and interacting with the Toll-like receptor (TLR) signaling pathway. However, the comprehensive role of the Notch signaling pathway in the innate immune response remains unknown. To assess the function of Notch1a in immunity, we examined the innate immune responses to Vibrio parahaemolyticus strain Vp13 of wild-type (WT) and notch1a-/- zebrafish larvae generated using the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system. The median lethal dose (LD50) of V. parahaemolyticus was significantly lower in notch1a-/- larvae than in WT larvae 3 days post fertilization (dpf). Transcriptome data analysis revealed 359 significantly differentially expressed genes (DEGs), including 246 significantly down-regulated genes and 113 significantly up-regulated genes, in WT infected groups compared with WT control groups. In contrast, 986 significantly DEGs were found in notch1a-/- infected groups compared with notch1a-/- control groups, of which 82 genes were significantly down-regulated and 904 genes were significantly up-regulated. These DEGs belonged to the tumor necrosis factor (TNF), complement, nuclear factor kappa B (NF-κB), cathepsin, interleukin (IL), chemokine, serpin peptidase inhibitor, matrix metallopeptidase, innate immune cells, pattern recognition receptor (PRR), and other cytokine families. Our results indicate that Notch1a plays roles in inhibiting many immunity-related genes and could comprehensively mediate the innate immune response by regulating TLRs, nucleotide-binding-oligomerization-domain-like receptors (NLRs), lectins, complement, ILs, chemokines, TNF, cathepsin, and serpin. Further studies are required to understand the specific mechanism of Notch1a in innate immunity.


Assuntos
Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Imunidade Inata/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Receptor Notch1/genética , Receptor Notch1/imunologia , Transdução de Sinais/imunologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Doenças dos Peixes/imunologia , Vibrioses/imunologia , Vibrioses/veterinária , Vibrio parahaemolyticus/fisiologia
7.
Emerg Microbes Infect ; 8(1): 1003-1016, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31282298

RESUMO

Zika virus (ZIKV) is a mosquito-borne Flavivirus that causes Zika disease with particular neurological complications, including Guillain-Barré Syndrome and congenital microcephaly. Although ZIKV has been shown to directly infect human neural progenitor cells (hNPCs), thereby decreasing their viability and growth, it is as yet unknown which of the cellular pathways involved in the disruption of neurogenesis are affected following ZIKV infection. By comparing the effect of two ZIKV strains in vitro on hNPCs, the differentiation process of the latter cells was found to lead to a decreased susceptibility to infection and cell death induced by each of the ZIKV strains, which was associated with an earlier and stronger antiviral innate immune response in infected, differentiated hNPCs, as compared to undifferentiated cells. Moreover, ZIKV modulated, both in hNPCs and in vivo in fetal brain in an experimental mouse model, the expression of the Notch pathway which is involved in cellular proliferation, apoptosis and differentiation during neurogenesis. These results show that the differentiation state of hNPCs is a significant factor contributing to the outcome of ZIKV infection and furthermore suggest that ZIKV infection might initiate early activation of the Notch pathway resulting in an abnormal differentiation process, implicated in ZIKV-induced brain injury.


Assuntos
Células-Tronco Neurais/virologia , Neurogênese , Receptor Notch1/metabolismo , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Apoptose , Feminino , Humanos , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor Notch1/genética , Transdução de Sinais , Zika virus/genética , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismo , Infecção por Zika virus/fisiopatologia
8.
Int J Mol Sci ; 20(8)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003475

RESUMO

Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multi-parametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice co-injected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients' blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro co-culturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs; and induced their pro-tumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bi-directional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immuno-suppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease.


Assuntos
Neoplasias da Mama/sangue , Carcinogênese/genética , Melanoma/sangue , Células Supressoras Mieloides/metabolismo , Adulto , Idoso , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transplante de Células/métodos , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Pessoa de Meia-Idade , Células Supressoras Mieloides/patologia , Fator 2 Relacionado a NF-E2/genética , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptor Notch1/genética , Proteínas de Transporte Vesicular/genética
9.
BMC Cancer ; 19(1): 391, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023264

RESUMO

BACKGROUND: Oral cancer has been estimated as the sixth most frequent solid cancer all over the world, in which tongue squamous cell carcinoma (TSCC) is the most common type of oral cancers. However, the mechanism of TSCC metastasizing to lymph node and distant sites has not been completely understood. METHODS: In this study, RT-qPCR method was used to detect the mRNA level of Numb, PTEN and Notch1 genes, as well as EMT-associated genes. Western blot assay was utilized to detect protein level of these genes. In addition, we determined cell proliferation by MTT assay and employed transwell invasion assay and wound healing assay to probe the abilities of invasion and migration, respectively. To investigate the role of PTEN, its inhibitor VO-Ohpic trihydrate was used to treat SCC-4 and CAL27 cells. RESULTS: We found that Numb expression was downregulated in SCC-9 and CAL-27 cells compared to NHOK cells. Instead, Notch1 level in SCC-9 and CAL-27 cells were higher than that in NHOK cells. Furthermore, the results showed that Numb overexpression significantly suppressed proliferation, migration and invasion of SCC-9 and CAL-27 cells via regulating Notch1 signaling and EMT-related genes expression. By contrast, we observed that RBP-Jκ knockdown had an inhibitory role in proliferation, migration and invasion of SCC-9 and CAL-27 cells. In cells with Numb overexpression or RBP-Jκ knockdown, p-FAK and EMT-related genes were remarkably regulated. CONCLUSIONS: Our findings provide new mechanism of understanding the metastasis of TSCC and help develop therapeutic strategies for treating tongue cancer.


Assuntos
Transição Epitelial-Mesenquimal/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Receptor Notch1/genética , Neoplasias da Língua/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , PTEN Fosfo-Hidrolase/genética , Transdução de Sinais/genética , Neoplasias da Língua/patologia
10.
Proc Natl Acad Sci U S A ; 116(14): 6985-6994, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30886104

RESUMO

Diabetic foot ulcerations (DFUs) represent a major medical, social, and economic problem. Therapeutic options are restricted due to a poor understanding of the pathogenic mechanisms. The Notch pathway plays a pivotal role in cell differentiation, proliferation, and angiogenesis, processes that are profoundly disturbed in diabetic wounds. Notch signaling is activated upon interactions between membrane-bound Notch receptors (Notch 1-4) and ligands (Jagged 1-2 and Delta-like 1, 3, 4), resulting in cell-context-dependent outputs. Here, we report that Notch1 signaling is activated by hyperglycemia in diabetic skin and specifically impairs wound healing in diabetes. Local inhibition of Notch1 signaling in experimental wounds markedly improves healing exclusively in diabetic, but not in nondiabetic, animals. Mechanistically, high glucose levels activate a specific positive Delta-like 4 (Dll4)-Notch1 feedback loop. Using loss-of-function genetic approaches, we demonstrate that Notch1 inactivation in keratinocytes is sufficient to cancel the repressive effects of the Dll4-Notch1 loop on wound healing in diabetes, thus making Notch1 signaling an attractive locally therapeutic target for the treatment of DFUs.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Pé Diabético/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Cicatrização , Idoso , Animais , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Pé Diabético/genética , Pé Diabético/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Receptor Notch1/genética
11.
J Med Microbiol ; 68(4): 649-656, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30843783

RESUMO

BACKGROUND: Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. Infection of RSV has been shown to induce Th lymphocyte differentiation. The present study was designed to determine the effects of RSV on the expression of Notch-1 and the related mechanisms on subsequent differentiation of Th lymphocytes. METHODS: A RSV-infected animal model was established and investigated at 7, 28 and 60 days post infection. Real-time qPCR and Western blot were used to observe the expression levels of Notch-1 in CD4+ T cells and its five ligands in lung tissues. The methylation levels of CpG islands in autoimmune regulator (AIRE) and Notch-1 promoters were analysed by time-of-flight mass spectrometry. The differentiation of Th lymphocytes was assayed by real-time qPCR. The distribution of JAG1 and DLL3 in the lung tissues were assayed by immunohistochemistry. The correlation between Th17 and DLL3 was analysed by simple correlation. RESULTS: The results showed that RSV promoted the expression and de-methylation of Notch-1 promoters in CD4+ T cells. Moreover, RSV infection promoted Th1 differentiation at day 7 and day 28; Th17 differentiation at day 7, day 28 and day 60; Th2 differentiation at day 28 and day 60. At the same time, RSV infection promoted the expression of JAG1 and DLL3. Activation of Notch-1/ DLL 3 in lungs may be associated with the differentiation of Th17 lymphocytes. CONCLUSIONS: Our data showed that activation of RSV stimulated the differentiation of Th17 in airway microenvironment through activation Notch-1/DLL3, which may be associated with the occurrence and development of RSV-induced asthma.


Assuntos
Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/imunologia , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Vírus Sincicial Respiratório Humano/imunologia , Células Th17/citologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/virologia , Linfócitos T CD4-Positivos , Microambiente Celular/imunologia , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Pulmão/virologia , Masculino , Proteínas de Membrana/genética , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch1/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Células Th1/imunologia , Células Th17/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Mol Med Rep ; 19(4): 3353-3360, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816513

RESUMO

Resveratrol, a natural polyphenol compound, has been reported to exert anticancer activity in various cancer cells. The present study investigated the effect and underlying mechanisms of resveratrol in the human ovarian cancer cell lines, A2780 and SKOV3. Treatment with resveratrol induced apoptotic cell death in dose­ and time­dependent manners, as well as a transient increase of reactive oxygen species (ROS) generation. Resveratrol­induced cell death was attenuated by the antioxidant, N­acetylcysteine (NAC), suggesting that ROS were involved in the observed cell death. Treatment with resveratrol resulted in a ROS­dependent decrease of Notch1 signaling. When cells were transfected to overexpress Notch1 using EF.hlCN1.CMV.GFP, resveratrol­induced cell death was blocked. Western blot analysis demonstrated that resveratrol also upregulated phospho­phosphatase and tensin homolog (p­PTEN) and downregulated phospho­Akt (p­Akt). Overexpression of p­Akt by transfection with a constitutively active form (caAkt), and the p­PTEN inhibitor SF1670 prevented resveratrol­induced cell death. The caspase­3 inhibitor z­DEVD­FMK significantly attenuated the resveratrol­induced caspase­3 cleavage. Taken together, the results of the present study suggest that resveratrol induces caspase­dependent cell death through suppression of Notch1 and PTEN/Akt signaling and it is mediated by increased ROS generation in human ovarian cancer cells.


Assuntos
Neoplasias Ovarianas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor Notch1/metabolismo , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptor Notch1/genética
13.
Nanoscale ; 11(14): 6693-6709, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30900717

RESUMO

Although nitric oxide (NO) can be used to treat osteoarthritis (OA) by inhibiting inflammation, a method for the accurately controlled release of NO in inflammatory cells is still elusive. Herein, photothermal-triggered NO nanogenerators NO-Hb@siRNA@PLGA-PEG (NHsPP) were constructed by assembling photothermal-agents and NO molecules within nanoparticles. In the NHsPP nanoparticles the hemoglobin (Hb) nanoparticles can act as a NO carrier which can absorb near-infrared light at 650 nm (0.5 W cm-2) and convert it into heat to trigger the release of NO. Moreover, after loading Notch1-siRNA, precise treatment can be achieved. Furthermore, using the synergistic effect of photothermal therapy, the NHsPP nanoparticles achieved simultaneous treatment with NO, siRNA and PTT. Through this combination therapy, the therapeutic effect of the NHsPP nanoparticles was significantly enhanced compared to the treatment groups using only NO, siRNA or PTT. This combination therapy inhibits the inflammatory response effectively by reducing the level of pro-inflammatory cytokines and the macrophage response. Subsequently, guided by dual-modal imaging, the NHsPP nanoparticles can not only accumulate effectively in OA mice, but can also reduce the inflammatory response and efficiently prevent cartilage erosion, without causing toxic side effects in the major organs. Therefore, this novel photothermal nanoparticle-based NO-releasing system is expected to be a potential alternative for clinical inflammatory disease therapy and may provide image guidance when combined with other nanotherapy systems.


Assuntos
Nanopartículas/química , Óxido Nítrico/química , Osteoartrite/terapia , RNA Interferente Pequeno/química , Animais , Cartilagem/patologia , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Citocinas/metabolismo , Feminino , Hemoglobina A Glicada/química , Humanos , Raios Infravermelhos , Articulações/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Óxido Nítrico/farmacologia , Óxido Nítrico/uso terapêutico , Osteoartrite/patologia , Fototerapia , Polietilenoglicóis/química , Poliglactina 910/química , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Receptor Notch1/metabolismo
14.
Gastroenterology ; 157(1): 193-209.e9, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30872106

RESUMO

BACKGROUND & AIMS: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes. METHODS: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE-/-) or peptidyl arginine deiminase type IV (Pad4-/-) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1iΔEC). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice. RESULTS: Expression of the neutrophil chemoattractant CXCL1 was up-regulated in primary LSECs exposed to mechanical stretch, compared with unexposed cells. Intravital imaging of livers in control mice revealed sinusoidal complexes of neutrophils and platelets and formation of NETs after pIVCL. NE-/- and Pad4-/- mice had lower portal pressure and livers had less fibrin compared with control mice after pIVCL and bile-duct ligation; neutrophil recruitment into sinusoidal lumen of liver might increase portal pressure by promoting sinusoid microthrombi. RNA-sequencing of LSECs identified proteins in mechanosensitive signaling pathways that are altered in response to mechanical stretch, including integrins, Notch1, and calcium signaling pathways. Mechanical stretch of LSECs increased expression of CXCL1 via integrin-dependent activation of transcription factors regulated by Notch and its interaction with the mechanosensitive piezo calcium channel. CONCLUSIONS: In studies of LSECs and knockout mice, we identified mechanosensitive angiocrine signals released by LSECs which promote PHTN by recruiting sinusoidal neutrophils and promoting formation of NETs and microthrombi. Strategies to target these pathways might be developed for treatment of PHTN. RNA-sequencing accession number: GSE119547.


Assuntos
Capilares/metabolismo , Quimiocina CXCL1/metabolismo , Células Endoteliais/metabolismo , Hipertensão Portal/metabolismo , Fígado/irrigação sanguínea , Infiltração de Neutrófilos , Estresse Mecânico , Trombose/metabolismo , Animais , Sinalização do Cálcio , Capilares/citologia , Armadilhas Extracelulares , Hidrolases/genética , Técnicas In Vitro , Integrinas/metabolismo , Elastase de Leucócito/genética , Ligadura , Fígado/metabolismo , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pressão na Veia Porta , Receptor Notch1/genética , Veia Cava Inferior/cirurgia
15.
DNA Cell Biol ; 38(4): 341-351, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30839226

RESUMO

Metastasis of ovarian cancer is regulated by microRNAs. This study focused on the effects of miR-30a-5p on ovarian cancer migration and invasion. Our results showed that the miR-30a-5p and mucin type O-glycan biosynthesis are closely related to ovarian cancer, and that miR-30a-5p was downregulated in ovarian cancer cells. miR-30a-5p overexpression reduced cell viability and inhibited migration and invasion in HO-8910 and HO-8910PM cells. S phase kinase-associated protein 2 (SKP2), B cell lymphoma 9 (BCL9), and NOTHC1 are direct target genes of miR-30a-5p. MTDH, SKP2, BCL9, and NOTCH1 genes were overexpressed in ovarian cancer cells, and they are direct target genes of miR-30a-5p. miR-30a-5p overexpression inhibited epithelial-mesenchymal transition (EMT) process, while upregulation of SKP2, BCL9, and NOTCH1 gene expression levels reduced the inhibition of EMT process by miR-30a-5p. miR-30a-5p was lowly expressed in ovarian cancer, and such a phenomenon is related to ovarian cancer metastasis. miR-30a-5p might inhibit the migration and invasion of ovarian cancer cells by downregulating the expression of SKP2, BCL9, and NOTCH1 genes.


Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Ovarianas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Biologia Computacional , Feminino , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Receptor Notch1/genética , Proteínas Quinases Associadas a Fase S/genética
16.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837354

RESUMO

To examine the protective effect of transplanting bone marrow mesenchymal stem cells (BMSCs) in treating lung injuryinduced by smoke exposure and to investigate the underlying mechanisms of this protection. SD rats were randomlydivided into four groups: normal group, normal +BMSCGFP group, smoke group, and smoke +BMSCGFP group. Todetect lung injury, we measured arterial blood gas, the wet-to-dry weight ratio, and levels of interleukin-1b, tumor necrosisfactor-a, interleukin-10, and interleukin-13 in bronchoalveolar lavage fluid and lung tissues. We also conductedhistopathology examinations. The protein markers of alveolar epithelial cells were measured to determine the BMSCdifferentiation. The protein levels of Notch1, Jagged-1, and Hes-1 also were detected. In the present study, BMSCtransplantation significantly decreased the wet-dry weight ratio of the lung, reduced the production of inflammatorymediators, and alleviated lung injury simply through differentiating into alveolar type II cells and alveolar type I cells. Western blot analysis confirmed that the protein expression of Notch-1, Jagged-1, and Hes-1 increased significantly aftersystemic BMSC transplantation. No significant difference was observed between the normal group and the nor-mal +BMSCGFP group. Our findings indicate that systemic transplantation of BMSCs alleviated lung injury induced bysmoke exposure, which may be associated with BMSCs' ability to differentiate into alveolar-type cells via the Notchsignaling pathway.


Assuntos
Proteína Jagged-1/genética , Transplante de Células-Tronco Mesenquimais , Receptor Notch1/genética , Lesão por Inalação de Fumaça/terapia , Fatores de Transcrição HES-1/genética , Células Epiteliais Alveolares/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Humanos , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Interleucina-1beta/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Notch/genética , Transdução de Sinais/genética , Lesão por Inalação de Fumaça/genética , Lesão por Inalação de Fumaça/patologia
17.
Biochimie ; 160: 193-199, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30905732

RESUMO

Ovarian cancer is one the prevalent cancers in women and is responsible for 5% of all the cancer related mortalities in women. Owing to late diagnosis, frequent relapses, side effects of chemotherapy, development of drug resistance, there is pressing need to screen out novel and effective treatment options. Accumulating evidences suggest that miRNAs may prove essential therapeutic targets for the treatment of cancer. This study was designed to investigate the role and therapeutic potential of miR-34 in ovarian cancer. It was found that miR-34 is significantly downregulated in ovarian cancer cell lines. Overexpression of miR-34 causes significant decrease in the proliferation of OVACAR-3 ovarian cancer cells via activation of apoptosis and autophagy. The miR-34 overexpression was concomitant with upsurge of apoptosis related proteins (Bax) and the autophagy associated protein (LC3 II and p62). TargetScan analysis showed Notch 1 to be the main target of miR-34 in OVACAR-3 cells which was further validated by luciferase reporter assay. The qRT-PCR results showed Notch 1 to be 3.2-4.1 fold higher in the ovarian cancer cell lines relative to the non-cancerous cells. Nonetheless, miR-34 overexpression in OVACAR-3 cells resulted in the post-transcriptional suppression of Notch 1 expression. Silencing of Notch 1 also caused inhibition of OVACAR-3 cell proliferation via induction of apoptosis and autophagy. Overexpression of Notch 1 could partially rescue the effects of miR-34 overexpression on the proliferation of OVACAR-3 cells. Moreover, overexpression of miR-34 causes significant inhibition of the invasion of the OVACAR-3 cells. The findings of the present study indicate the tumor suppressive role of miR-34 in ovarian cancer and may therefore prove to be a potential therapeutic target.


Assuntos
Apoptose , Autofagia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Ovarianas/patologia , Receptor Notch1/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Receptor Notch1/genética , Células Tumorais Cultivadas
20.
Cancer Sci ; 110(5): 1644-1652, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30784169

RESUMO

Cancer tissues contain small populations of highly tumorigenic cells termed cancer stem cells (CSCs). Immortalized cell lines containing CSCs are valuable and powerful experimental tools for research into the characteristics of these stem cells. We previously reported that the hepatocellular carcinoma cell line Li-7 includes abundant CD13+ CD166- CSCs; however, the number of these cells decreases after long-term culture as a result of differentiation to non-CSC populations. To ensure consistent and reproducible results in experiments using Li-7 cells, it is important that the CSC population is maintained stably regardless of culture duration and passage. In the present study, we found that a commercially available culture medium for maintenance of embryonic stem cells and induced pluripotent stem cells, mTeSR1, effectively prevented spontaneous differentiation by CD13+ CD166- cells to CD13- CD166+ cells and therefore maintained the CSC population in Li-7 cell cultures. CD13+ CD166- CSCs maintained using this culture medium retained high tumorigenicity after transplantation into mice; they also showed the ability to differentiate in vitro into non-CSC populations in RPMI-1640 with 10% FBS medium. We analyzed gene expression profiles of CSC and non-CSC populations in Li-7 cultures using an RNA sequencing method. Genes such as FGFR, NOTCH1, and JAG1, that are associated with tumorigenicity and stemness, were upregulated in the CSC population. Our results suggest that CSCs can be maintained in immortalized cancer cell lines cultured over an extended period using a medium developed for culture of embryonic/induced pluripotent stem cells.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultura de Células/métodos , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/transplante , Animais , Antígenos CD/metabolismo , Antígenos CD13/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular Neuronais/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura/farmacologia , Proteínas Fetais/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteína Jagged-1/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Receptor Notch1/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Análise de Sequência de RNA , Regulação para Cima
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