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1.
Molecules ; 24(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060229

RESUMO

Background: KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. Methods: In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). Results: we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. Conclusions: These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.


Assuntos
Neoplasias da Mama/enzimologia , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas Nucleares/genética , Radiossensibilizantes/farmacologia , Proteínas Repressoras/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/química , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/química , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiação
2.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083348

RESUMO

Detrimental health consequences from exposure to space radiation are a major concern for long-duration human exploration missions to the Moon or Mars. Cellular responses to radiation are expected to be heterogeneous for space radiation exposure, where only high-energy protons and other particles traverse a fraction of the cells. Therefore, assessing DNA damage and DNA damage response in individual cells is crucial in understanding the mechanisms by which cells respond to different particle types and energies in space. In this project, we identified a cell-specific signature for radiation response by using single-cell transcriptomics of human lymphocyte subpopulations. We investigated gene expression in individual human T lymphocytes 3 h after ex vivo exposure to 2-Gy gamma rays while using the single-cell sequencing technique (10X Genomics). In the process, RNA was isolated from ~700 irradiated and ~700 non-irradiated control cells, and then sequenced with ~50 k reads/cell. RNA in each of the cells was distinctively barcoded prior to extraction to allow for quantification for individual cells. Principal component and clustering analysis of the unique molecular identifier (UMI) counts classified the cells into three groups or sub-types, which correspond to CD4+, naïve, and CD8+/NK cells. Gene expression changes after radiation exposure were evaluated using negative binomial regression. On average, BBC3, PCNA, and other TP53 related genes that are known to respond to radiation in human T cells showed increased activation. While most of the TP53 responsive genes were upregulated in all groups of cells, the expressions of IRF1, STAT1, and BATF were only upregulated in the CD4+ and naïve groups, but were unchanged in the CD8+/NK group, which suggests that the interferon-gamma pathway does not respond to radiation in CD8+/NK cells. Thus, single-cell RNA sequencing technique was useful for simultaneously identifying the expression of a set of genes in individual cells and T lymphocyte subpopulation after gamma radiation exposure. The degree of dependence of UMI counts between pairs of upregulated genes was also evaluated to construct a similarity matrix for cluster analysis. The cluster analysis identified a group of TP53-responsive genes and a group of genes that are involved in the interferon gamma pathway, which demonstrate the potential of this method for identifying previously unknown groups of genes with similar expression patterns.


Assuntos
Exposição à Radiação , Fator de Transcrição STAT1/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Análise por Conglomerados , Raios gama , Humanos , Imunofenotipagem , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
3.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096691

RESUMO

Blue light (BL) plays an important role in regulation of the growth and development of aquatic plants and land plants. Aureochrome (AUREO), the recent BL photoreceptor identified in photosynthetic stramenopile algae, is involved in the photomorphogenesis and early development of Saccharina japonica porophytes (kelp). However the factors that interact with the SjAUREO under BL conditions specifically are not clear. Here in our study, three high quality cDNA libraries with CFU over 5 × 106 and a recombination rate of 100% were constructed respectively through white light (WL), BL and darkness (DK) treatments to the juvenile sporophytes. Based on the constructed cDNA libraries, the interactors of SjAUREO were screened and analyzed. There are eighty-four genes encoding the sixteen predicted proteins from the BL cDNA library, sixty-eight genes encoding eighteen predicted proteins from the DK cDNA library, and seventy-four genes encoding nineteen proteins from the WL cDNA library. All the predicted proteins are presumed to interact with SjAUREO when co-expressed with SjAUREO seperately. The 40S ribosomal protein S6 (RPS6), which only exists in the BL treated cDNA library except for two other libraries, and which is essential for cell proliferation and is involved in cell cycle progression, was selected for detailed analysis. We showed that its transcription was up-regulated by BL, and was highly transcribed in the basal blade (meristem region) of juvenile sporophytes but less in the distal part. Taken together, our results indicated that RPS6 was highly involved in BL-mediated kelp cellular division and photomorphogenesis by interacting with SjAUREO.


Assuntos
Laminaria/metabolismo , Laminaria/efeitos da radiação , Luz , Proteína S6 Ribossômica/metabolismo , Proteína S6 Ribossômica/efeitos da radiação , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/efeitos da radiação , Proliferação de Células , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Biblioteca Gênica , Genes de Plantas/genética , Laminaria/genética , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efeitos da radiação , Fotossíntese , Proteínas de Plantas/genética , Proteínas Ribossômicas/genética , Regulação para Cima/efeitos da radiação
4.
Int J Oncol ; 54(4): 1466-1480, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30968148

RESUMO

It is well-known that the activation status of the P53, signal transducer and activator of transcription (Stat)3 and nuclear factor (NF)­κB signaling pathways determines the radiosensitivity of cancer cells. However, the function of these pathways in radiosensitive vs radioresistant cancer cells remains elusive. The present study demonstrated that adaptive expression of epidermal growth factor (EGF) following exposure to ionizing radiation (IR) may induce radiosensitization of pancreatic cancer (PC) cells through induction of the cyclin D1/P53/poly(ADP­ribose) polymerase pathway. By contrast, adaptively expressed interleukin (IL)­6 and insulin­like growth factor (IGF)­1 may promote radioresistance of PC cells, likely through activation of the Stat3 and NF­κB pathways. In addition, cyclin D1 and survivin, which are specifically expressed in the G1/S and G2/M phase of the cell cycle, respectively, are mutually exclusive in radiosensitive and radioresistant PC cells, while Bcl­2 and Bcl­xL expression does not differ between radiosensitive and radioresistant PC cells. Therefore, adaptively expressed EGF and IL­6/IGF­1 may alter these pathways to promote the radiosensitivity of PC cancers. The findings of the present study highlight potential makers for the evaluation of radiosensitivity and enable the development of effective regimens for cancer radiotherapy.


Assuntos
Carcinoma Ductal Pancreático/radioterapia , Fator de Crescimento Epidérmico/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias Pancreáticas/radioterapia , Transdução de Sinais/efeitos da radiação , Apoptose/efeitos da radiação , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Humanos , Neoplasias Pancreáticas/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Tolerância a Radiação , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos da radiação
5.
Eur J Pharmacol ; 852: 134-141, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30831080

RESUMO

Ovarian cancer (OC) is a major cause of cancer-related deaths in women all over the world. The easy metastasis of OC and the problem of radioresistance are serious issues remaining to be overcome. Thus, research on molecular mechanisms underlying is in urgent demand. Long non-coding RNAs (lncRNAs) are a class of RNAs without protein coding potential, which has been reported to participate in the regulation on multiple biological process in cancers, including cell radiosensitivity and metastasis. Present study aimed to explore the role of lncRNA FAM83-AS1 in radioresistance and metastasis of ovarian cancer. First of all, the obvious upregulation of FAM83H-AS1 was identified by qRT-PCR in OC tissues, especially in metastatic tissues, as well as in OC cell lines. Importantly, we confirmed the correlation of FAM83H-AS1 levels with both ovarian cancer cells and normal ovarian cells. And Kaplan-Meier analysis indicated FAM83H-AS1 as a potential target of poor prognosis of OC. Through loss-of-function assays, we validated the inductive effect of FAM83H-AS1 in OC cell metastasis and radioresistance. Through mechanism research on FAM83H-AS1, we confirmed its interaction with HuR using pull-down assay and RNA immunoprecipitation (RIP), and verified the stabilization of HuR protein by FAM83-AS1 through western blot with the addition of CHX. Finally, rescue assays showed that overexpression of HuR rescued the suppression on radioresistance and metastasis in OC cell caused by the silencing of FAM83H-AS1. In conclusion, present study proved that FAM83H-AS1 contributes to the radioresistance and cell metastasis in ovarian cancer through stabilizing HuR protein.


Assuntos
Proteína Semelhante a ELAV 1/genética , Neoplasias Ovarianas/patologia , RNA Longo não Codificante/genética , Tolerância a Radiação/genética , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/efeitos da radiação , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Ovarianas/genética , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
6.
Int J Mol Sci ; 20(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925808

RESUMO

The roles of low-intensity pulsed ultrasound (LIPUS) and microRNAs (miRNAs) on hMSCs commitments have already been investigated; however, the effects of the application of their co-treatments in an in vitro cell model are still unknown. Our previous studies demonstrated that (i) LIPUS modulated hMSCs cytoskeletal organization and (ii) miRNA-675-5p have a role in HIF-1α signaling modulation during hMSCs osteoblast commitment. We investigated for the first time the role of LIPUS as promoter tool for miRNA expression. Thanks to bioinformatic analysis, we identified miR-31-5p as a LIPUS-induced miRNA and investigated its role through in vitro studies of gain and loss of function. Results highlighted that LIPUS stimulation induced a hypoxia adaptive cell response, which determines a reorganization of cell membrane and cytoskeleton proteins. MiR-31-5p gain and loss of function studies, demonstrated as miR-31-5p overexpression, were able to induce hypoxic and cytoskeletal responses. Moreover, the co-treatments LIPUS and miR-31-5p inhibitor abolished the hypoxic responses including angiogenesis and the expression of Rho family proteins. MiR-31-5p was identified as a LIPUS-mechanosensitive miRNAs and may be considered a new therapeutic option to promote or abolish hypoxic response and cytoskeletal organization on hMSCs during the bone regeneration process.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco Mesenquimais/efeitos da radiação , MicroRNAs/genética , Ondas Ultrassônicas , Regulação para Cima/efeitos da radiação , Diferenciação Celular , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia
7.
Prep Biochem Biotechnol ; 49(2): 184-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30712452

RESUMO

Microbial enzymes of extremophilic origin serve as a vital source of stable industrial enzymes. The present study focused on overproduction of a thermoalkalophilic lipase produced by Bacillus atrophaeus FSHM2 through UV-induced random mutagenesis (5-45 min exposure to UV light) and factorial experimental design augmented to response surface methodology. Firstly, a UV-induced mutant (designated as UV-45) was developed after the exposure of wild strain to UV irradiation for 45 min which was able to secrete 3484.8 U/L lipase. Afterward, Plackett-Burman experimental approach augmented to central composite design was employed to optimize medium components (olive oil, maltose, glucose, sucrose, yeast extract, tryptone, urea, (NH4)2SO4, NaCl, CaCl2, and ZnSO4) for lipase production by the UV-45 mutant strain. The maximum lipase production of 5505.3 U/L were predicted in medium containing 5% of olive oil, 0.69% of glucose, 0.69% of sucrose, 2.5% of maltose, yeast extract (0.7 g/L), urea (0.44 g/L), (NH4)2SO4 (2.44 g/L), tryptone (1.19 g/L), NaCl (1.61 g/L), CaCl2 (3.81 g/L), and ZnSO4 (1.42 g/L). A mean value of 5161.3 ± 83.3 U/L of lipolytic activity was acquired from real experiments. To sum up, the lipolytic activity of wild type strain (1720.4 U/L) increased by 3-fold after UV-induced mutagenesis and medium components optimization (5161.3 U/L).


Assuntos
Bacillus/genética , Bacillus/efeitos da radiação , Proteínas de Bactérias/genética , Lipase/genética , Mutagênese/efeitos da radiação , Regulação para Cima/efeitos da radiação , Bacillus/enzimologia , Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Microbiologia Industrial/métodos , Lipase/metabolismo , Mutação/efeitos da radiação , Raios Ultravioleta
8.
Med Sci Monit ; 25: 1214-1219, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30763293

RESUMO

BACKGROUND POU5F1B, serving as a carcinogen, participates in radiosensitivity of several tumors. However, in esophageal cancer, its potential mechanism and function in regulating radiosensitivity remain unclear. MATERIAL AND METHODS The expression level of POU5F1B was detected in plasma of esophageal tumor patients and cancer cell lines. The effect of POU5F1B knockdown on cell proliferation and colony formation was determined using CCK-8 assay and colony formation assay. Cell apoptosis rate was detected by flow cytometry. RESULTS POU5F1B expression level declined after radiotherapy in the plasma of esophageal cancer patients (p=0.025). Compared with HEEPIC, the level of POU5F1B was upregulated in ECA109 (p<0.01), ECA9706 (p<0.01), KYSE410 (p<0.01), and KYSE510 (p=0.036). The silencing of POU5F1B played a role in inhibiting colony formation. After radiotherapy, the apoptosis rates in the ECA109 with 4Gy si-POU5F1B group and 4Gy si-NC group were 39.1±0.1% and 35.3±0.1%, respectively (p=0.0193). The rate was 21.00±0.1 and 29.1±0.1% (p<0.0072) in the si-NC group and si-POU5F1B group, respectively. For proliferation rate, 4Gy si-POU5F1B ECA109 performed better than 4Gy si-NC. CONCLUSIONS Radiotherapy contributed to the decline in the expression level of POU5F1B in plasma, which was upregulated in ECA109, ECA9706, KYSE410, and KYSE510, but not in HEEPIC. The knockdown of POU5F1B increased the radiosensitivity of esophageal cancer cell lines.


Assuntos
Neoplasias Esofágicas/radioterapia , Fator 3 de Transcrição de Octâmero/deficiência , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Neoplasias Esofágicas/sangue , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Técnicas de Silenciamento de Genes/métodos , Genes myc , Humanos , Fator 3 de Transcrição de Octâmero/sangue , Fator 3 de Transcrição de Octâmero/genética , RNA Longo não Codificante/genética , Tolerância a Radiação , Regulação para Cima/efeitos da radiação
9.
J Cancer Res Clin Oncol ; 145(4): 881-893, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30701326

RESUMO

PURPOSE: Tumor cells generally exhibit higher levels of reactive oxygen species (ROS), however, when stressed, tumor cells can undergo a process of 'Redox Resetting' to acquire a new redox balance with stronger antioxidant systems that enable cancer cells to become resistant to radiation therapy (RT). Here, we describe how RT affects the oxidant/antioxidant balance in human embryonal (RD) and alveolar (RH30) rhabdomyosarcoma (RMS) cell lines, investigating on the molecular mechanisms involved. METHODS: Radiations were delivered using an x-6 MV photon linear accelerator and their effects were assessed by vitality and clonogenic assays. The expression of specific antioxidant-enzymes, such as Superoxide Dismutases (SODs), Catalase (CAT) and Glutathione Peroxidases 4 (GPx4), miRNAs (miR-22, -126, -210, -375, -146a, -34a) and the transcription factor NRF2 was analyzed by quantitative polymerase chain reaction (q-PCR) and western blotting. RNA interference experiments were performed to evaluate the role of NRF2. RESULTS: Doses of RT higher than 2 Gy significantly affected RMS clonogenic ability by increasing ROS production. RMS rapidly and efficiently brought back ROS levels by up-regulating the gene expression of antioxidant enzymes, miRNAs as well as of NRF2. Silencing of NRF2 restrained the RMS ability to counteract RT-induced ROS accumulation, antioxidant enzyme and miRNA expression and was able to increase the abundance of γ-H2AX, a biomarker of DNA damage, in RT-treated cells. CONCLUSIONS: Taken together, our data suggest the strategic role of oxidant/antioxidant balance in restraining the therapeutic efficiency of RT in RMS treatment and identify NRF2 as a new potential molecular target whose inhibition might represent a novel radiosensitizing therapeutic strategy for RMS clinical management.


Assuntos
Fator 2 Relacionado a NF-E2/metabolismo , Rabdomiossarcoma Alveolar/radioterapia , Rabdomiossarcoma Embrionário/radioterapia , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Humanos , MicroRNAs/biossíntese , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , Oxirredução/efeitos da radiação , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Tolerância a Radiação , Espécies Reativas de Oxigênio/metabolismo , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Embrionário/genética , Rabdomiossarcoma Embrionário/metabolismo , Transfecção , Regulação para Cima/efeitos da radiação
10.
J Radiat Res ; 60(3): 289-297, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30805606

RESUMO

Exosomes and other extracellular vesicles are key players in cell-to-cell communication, and it has been proposed that they are involved in different aspects of the response to ionizing radiation, including transmitting the radiation-induced bystander effect and mediating radioresistance. The functional role of exosomes depends on their molecular cargo, including proteome content. Here we aimed to establish the proteome profile of exosomes released in vitro by irradiated UM-SCC6 cells derived from human head-and-neck cancer and to identify processes associated with radiation-affected proteins. Exosomes and other small extracellular vesicles were purified by size-exclusion chromatography from cell culture media collected 24 h after irradiation of cells with a single 2, 4 or 8 Gy dose, and then proteins were identified using a shotgun LC-MS/MS approach. Exosome-specific proteins encoded by 1217 unique genes were identified. There were 472 proteins whose abundance in exosomes was significantly affected by radiation (at any dose), including 425 upregulated and 47 downregulated species. The largest group of proteins affected by radiation (369 species) included those with increased abundance at all radiation doses (≥2 Gy). Several gene ontology terms were associated with radiation-affected exosome proteins. Among overrepresented processes were those involved in the response to radiation, the metabolism of radical oxygen species, DNA repair, chromatin packaging, and protein folding. Hence, the protein content of exosomes released by irradiated cells indicates their actual role in mediating the response to ionizing radiation.


Assuntos
Vesículas Extracelulares/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Proteoma/metabolismo , Radiação Ionizante , Linhagem Celular Tumoral , Regulação para Baixo/efeitos da radiação , Exossomos/metabolismo , Vesículas Extracelulares/efeitos da radiação , Vesículas Extracelulares/ultraestrutura , Ontologia Genética , Humanos , Proteínas de Neoplasias/metabolismo , Regulação para Cima/efeitos da radiação
11.
Biochem Biophys Res Commun ; 509(2): 617-623, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30606477

RESUMO

Long non-coding RNAs (lncRNAs) play vital roles in the pathobiology of glioblastoma multiforme (GBM). Though radiotherapy remains the most effective component of multiple therapies for patients with GBM, lncRNAs conferring GBM radioresistance are less unknown. Here, the present study identified that the antisense transcript of hypoxia-inducible factor-1α (AHIF) was upregulated in GBM cells after radiotherapy. The deregulation of AHIF affected GBM cell clonogenic formation, DNA repair and apoptosis. Notably, knockdown of AHIF inhibited tumorigenesis after radiotherapy in vivo. Further biochemical analysis identified that AHIF regulated proteins associated with apoptosis after radiotherapy. Thus, the present data illustrate that suppression of AHIF increases radiosensitivity in GBM cells, which may be a potential diagnostic and therapeutic target for GBM patients.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/radioterapia , Glioblastoma/genética , Glioblastoma/radioterapia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , RNA Longo não Codificante/genética , Regulação para Cima , Animais , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Camundongos , Tolerância a Radiação , Regulação para Cima/efeitos da radiação
12.
Cells ; 8(1)2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30669263

RESUMO

Blue light is a major component of visible light and digital displays. Over-exposure to blue light could cause retinal damage. However, the mechanism of its damage is not well defined. Here, we demonstrate that blue light (900 lux) impairs cell viability and induces cell apoptosis in retinal neurocytes in vitro. A DNA electrophoresis assay shows severe DNA damage in retinal neurocytes at 2 h after blue light treatment. γ-H2AX foci, a specific marker of DNA double-strand breaks (DSBs), is mainly located in the Map2-posotive neuron other than the glia cell. After assaying the expression level of proteins related to DNA repair, Mre11, Ligase IV and Ku80, we find that Ku80 is up-regulated in retinal neurocytes after blue light treatment. Interestingly, Ku80 is mainly expressed in glia fibrillary acidic protein (GFAP)-positive glia cells. Moreover, following blue light exposure in vivo, DNA DSBs are shown in the ganglion cell layer and only observed in Map2-positive cells. Furthermore, long-term blue light exposure significantly thinned the retina in vivo. Our findings demonstrate that blue light induces DNA DSBs in retinal neurons, and the damage is more pronounced compared to glia cells. Thus, this study provides new insights into the mechanisms of the effect of blue light on the retina.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Luz , Neuroglia/patologia , Neuroglia/efeitos da radiação , Neurônios Retinianos/patologia , Neurônios Retinianos/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Autoantígeno Ku/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
13.
Oncol Rep ; 41(3): 1960-1970, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30569171

RESUMO

Radiotherapy (RT) is a traditional and important treatment for carcinoma of the esophagus along with surgery and chemotherapy. High mobility group box 1 (HMGB1) plays a crucial part in inhibiting the apoptosis of cancer cells after irradiation treatment. The present study, was designed to analyze the function of HMGB1 in esophageal cancer progression and elucidate the effects of HMGB1 on the radiosensitivity of human esophageal cancer cell lines. In the present study, an immunohistochemical evaluation of HMGB1 was performed on 77 biopsies, and the results revealed that HMGB1 overexpression was positively correlated with gross tumor volume (GTV), tumor­node­metastasis (TNM) stage, T classification, distant metastasis, and relapse and negatively correlated with patient survival rates, suggesting that HMGB1 acts as a key factor in the development of esophageal cancer. An shRNA targeting HMGB1 was designed for the knockdown of HMGB1 in ECA109 and TE13 cells, and the transfection efficiency of the shRNA was assessed using quantitative real­time reverse transcription polymerase chain reaction and western blot analysis. CCK­8 and clonogenic assays were used to analyze the effect of HMGB1 on the proliferation and radiosensitivity, respectively, of esophageal cancer cells in vitro. The influence of HMGB1 on radiation­induced changes in the migration, invasion, and cell cycle as well as apoptosis of tumor cells was examined by wound­healing and Transwell assays and flow cytometry, respectively. In addition, xenograft tumor models were constructed to observe the effect of HMGB1 on tumor growth in vivo. The results of the study in vitro revealed that the proliferation of the HMGB1­shRNA group decreased after irradiation, and the radiation treatment reduced the tumor volume of the xenograft model which was more marked in HMGB1­shRNA group. Moreover, HMGB1 was involved in the phosphorylation of H2AX after irradiation, and HMGB1 knockdown blocked the cell cycle in the G0/G1 phase and increased apoptosis. HMGB1 deficiency was also correlated with the upregulation of p16, Bax and caspase­9 and the downregulation of MMP­2, MMP­9, cyclin D1, CDK4, γH2AX and Bcl­2. These data indicated that the overexpression of HMGB1 prior to treatment was correlated with poor clinical outcome in esophageal carcinoma and that knockdown HMGB1 expression in human esophageal cancer cell lines increased their radiosensitivity by allowing the induction of apoptosis and G0/G1 arrest after exposure to radiation.


Assuntos
Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Proteína HMGB1/metabolismo , Tolerância a Radiação/genética , Idoso , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/radioterapia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/mortalidade , Carcinoma de Células Escamosas do Esôfago/radioterapia , Esôfago/patologia , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteína HMGB1/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , RNA Interferente Pequeno/metabolismo , Taxa de Sobrevida , Regulação para Cima/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Cell Physiol Biochem ; 50(5): 1929-1944, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30396174

RESUMO

BACKGROUND/AIMS: Nasopharyngeal carcinoma (NPC) is rare worldwide but remains highly prevalent in endemic regions, notably in southern China. Radiotherapy remains the treatment of choice for NPC, but radioresistance has been identified as a major cause of therapeutic failure. The Wnt/ß-catenin signaling has been found to be involved in NPC radioresistance; however, the effect of ß-catenin overexpression on radioresistance remains unknown in NPC until now. This study aimed to examine the impact of ß-catenin overexpression on the radiosensitivity of human NPC CNE-2 cells. METHODS: Immunohistochemistry was performed to detect the ß-catenin expression in normal nasopharyngeal specimens and NPC specimens. The human NPC CNE-2 cell line overexpressing ß-catenin was modeled by transfection with the pcDNA3.1/Hygro(+)/ß-catenin recombinant vector (transfection group), while cells transfected with the pcDNA3.1/Hygro(+) vector served as negative controls and non-transfected cells served as blank controls. The expression of key molecules of the Wnt/ß-catenin signaling pathway was determined using Western blotting and qPCR assays, and the changes of radiation sensitivity were measured with a colony-formation assay. Cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) assay. In addition, the cell cycle and apoptosis was detected using flow cytometry and the TCF/LEF transcriptional activity was measured with a Dual Luciferase Reporter Assay System. RESULTS: Immunohistochemical staining showed high ß-catenin expression in radioresistant NPC specimens, and low expression in radiosensitive NPC specimens and normal nasopharyngeal specimens. Western blotting and qPCR assays detected higher ß-catenin expression in the transfection group than in the negative and blank controls (P < 0.01). Down-regulation of GSK-3ß expression (P < 0.05) and up-regulation of Cyclin D1 expression (P < 0.01) was detected in ß-catenin overexpressing NPC cells exposed to X-ray radiation relative to negative and blank controls. Colony-formation assay revealed higher D0, Dq and SF in the transfection group than in the negative and blank control groups post-radiation, and the SER in the transfection group was 0.75-fold and 0.68-fold greater than that in the blank and negative control groups, respectively. MTT assay revealed that the viability of CNE-2 cells was significantly higher in the transfection group (96% ± 8.72%) than in the negative control group (74.67 ± 7.05%) and the blank control group (75.33% ± 7.02%) 24 h post-exposure to 6 Gy X-ray radiation (P < 0.05). X-ray radiation led to a lower proportion of CNE-2 cells at the G2/M phase and a lower apoptotic rate in the transfection group than in the negative and blank control groups (P < 0.05). In addition, the TCF/LEF transcriptional activity was higher in the transfection group than in the negative and blank control groups (P < 0.01), and 6 Gy X-ray radiation elevated the TCF/LEF transcriptional activity relative to 0 Gy radiation in the transfection group (P < 0.01). CONCLUSION: ß-catenin overexpression may decrease the radiation sensitivity in NPC CNE-2 cells through activating the downstream transcriptional factors of ß-catenin, and reducing G2/M arrest and cell apoptosis.


Assuntos
Carcinoma/patologia , Neoplasias Nasofaríngeas/patologia , Tolerância a Radiação , beta Catenina/metabolismo , Carcinoma/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Ciclina D1/metabolismo , Regulação para Baixo/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Regulação para Cima/efeitos da radiação , Via de Sinalização Wnt/efeitos da radiação , Raios X , beta Catenina/genética
15.
Chem Biol Interact ; 294: 144-150, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30125552

RESUMO

Currently, nanoparticles are used in various commercial products. One of the most common nanoparticles is titanium dioxide (TiO2). It has a catalytic activity and UV absorption, and generates reactive oxygen species (ROS). This catalytic activity of TiO2 nanoparticles was believed to be capable of killing a wide range of microorganisms. In the environment, the unique properties of TiO2 nanoparticles can be maintained; therefore, the increasing use of TiO2 nanoparticles is raising concerns about their environmental risks. Thus, assessment of the biological and ecological effects of TiO2-NOAAs is necessary. In this study, we assessed the effect of TiO2-NOAAs for S. cerevisiae using DNA microarray. To compare yeast cells under various conditions, six treatment conditions were prepared (1. adsorbed fraction to TiO2-NOAA under UV; 2. non-adsorbed fraction to TiO2-NOAA under UV; 3. adsorbed fraction to TiO2-NOAA without UV; 4. non-adsorbed fraction to TiO2-NOAA without UV; 5. under UV; and 6. untreated control). The result of the DNA microarray analysis, suggested that yeast cells that are adsorbed by TiO2-NOAA under UV irradiation suffer oxidative stress and this stress response was similar to that by only UV irradiation. We concluded that the effect of TiO2-NOAAs on yeast cells under UV irradiation is not caused by TiO2-NOAA but UV irradiation.


Assuntos
Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Titânio/química , Raios Ultravioleta , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Metabolismo Energético/genética , Nanopartículas Metálicas/química , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , Estresse Oxidativo/efeitos da radiação , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
16.
J Photochem Photobiol B ; 186: 69-80, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30015062

RESUMO

The awareness of the interrelationship between immunosenescence and constant light exposure can provide new insights into the consequences of excessive exposure to light at night due to light pollution or shift work. Here, we investigated whether constant light exposure (LL) acts as an inducer of immunosenescence. We also determined the role of melatonin or turmeric in reversing the putative effects of constant light and explored for the first time the underlying molecular mechanisms. Young (3-4-month-old) rats were exposed daily to LL alone or in combination with each of melatonin and turmeric for 12 weeks. A group of aged rats (18-months old; n = 6) was used as a reference for natural immunosenescence. Constant light exposure resulted in remarkable pathophysiological alterations resembling those noticed in normal aged rats, manifested as apparent decreases in antioxidant activities as well as Nrf2 and DJ-1 expressions, striking augmentation in oxidative stress, proinflammatory cytokines and expression of TNFα, Bax, and p53 genes, and deleterious changes of lymphoid organs, Co-administration of melatonin or turmeric was able to reverse all alterations induced by LL through upregulation of Nrf2/DJ-1 and downregulation of p53/Bax pathways. These data suggest that LL accelerates immunosenescence via oxidative stress and apoptotic pathways. They also demonstrate for the first time that turmeric is comparable to melatonin in boosting the immune function and counteracting the LL-associated immunosenescence. These effects suggest that turmeric supplementation can be used as an inexpensive intervention to prevent circadian disruption-related immunosenescence. However, to validate the effects of turmeric on humans further studies are warranted.


Assuntos
Imunossenescência/efeitos dos fármacos , Luz , Melatonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/efeitos da radiação , Citocinas/sangue , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Imunossenescência/efeitos da radiação , Masculino , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Oxirredutases/metabolismo , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Ratos , Transdução de Sinais/efeitos da radiação , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Timo/efeitos dos fármacos , Timo/metabolismo , Timo/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiação , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
17.
J Photochem Photobiol B ; 186: 31-40, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30005204

RESUMO

Oxidative stress, in which the amount of oxidants exceeds the capacity of antioxidant defense system, is a well-accepted pathogenesis of several human diseases. Light-emitting diode irradiation (LEDI) is an efficient strategy to counteract this condition. The biological effect of phototherapy, using visible light, has attracted recent attention especially in dermatological practice. However, little is known about the molecular mechanism of the anti-oxidant and anti-inflammatory effects of red light irradiation. We evaluated these effects of LEDI in HaCaT human keratinocyte cells under phorbol-12-myristate-13-acetate (PMA) induced reactive oxygen species (ROS). Microarray analysis revealed changes in 309 genes after LEDI. LEDI at 625 nm produced ROS scavenging and anti-inflammatory effects. One of the most important genes identified by microarray analysis was sphingosine kinase-1 (SPHK1), which is a key molecule in sphingolipid metabolism. SPHK1 knock-down drastically reduced ROS scavenging efficiency as well as expression levels of inflammation-related proteins in PMA-treated HaCaT cells. These results not only indicate the potential for the clinical application of 625-nm LEDI in treating skin disorders via ROS and/or inflammation, but also suggest SPHK1 as a potential therapeutic target in phototherapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Luz , NF-kappa B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/patologia , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Microscopia de Fluorescência , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiação
18.
J Photochem Photobiol B ; 186: 152-159, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30048845

RESUMO

Burn wound is a complex multi-factorial pathophysiology producing excruciating pain and psychological discomfort among patients, which imposes a major burden on the healthcare system. Multi-target therapy focuses on augmented healing by regulating different phases of tissue repair. Recently, photobiomodulation (PBM)-induced wound healing has achieved profound impetus as a non-invasive, drug-free biophysical therapeutic approach. On the other hand, medicinal honey known to possess antibacterial and immunomodulatory properties and is being used as an effective treatment option for infected wounds. The present study aimed to determine whether the combination of medicinal honey and PBM using superpulsed 904 nm laser treatment could additively accelerate full-thickness burn wound repair in rats. Animals were randomly allocated into 4 experimental groups: control (C), PBM superpulsed 904 nm laser treated (PBMT), honey treated (HT) and combined treatment (CT). The dual treatment exhibited an enhanced wound area contraction and hexosamine content as compared to the other groups. Histopathological analysis revealed increased cellular proliferation, extracellular matrix accumulation and decreased inflammation in the CT group. Further, the CT group demonstrated synergistically attenuated inflammation, pain and enhanced cell adhesion, migration as evidenced by significantly reduced protein expression of TNF-α, NF-κB, IL-1ß, COX-2, substance-P receptor and up-regulation of fibronectin, respectively as compared with the other groups. Thus, the findings of present study signify that the combination of medicinal honey and PBMT accelerates the repair process of burn wounds. The study showed that therapeutic efficacy of 904 nm superpulsed laser-mediated PBM augments in the presence of medicinal honey by enhancing cellular proliferation and attenuation of inflammation and pain in burn wound healing.


Assuntos
Mel , Inflamação , Lasers , Dor/prevenção & controle , Cicatrização/efeitos da radiação , Animais , Queimaduras/patologia , Queimaduras/radioterapia , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/efeitos da radiação , Hexosaminas/metabolismo , Inflamação/prevenção & controle , Interleucina-1beta/metabolismo , Terapia com Luz de Baixa Intensidade , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Dor/patologia , Ratos , Ratos Sprague-Dawley , Pele/metabolismo , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiação , Cicatrização/efeitos dos fármacos
19.
Anticancer Res ; 38(6): 3323-3331, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29848680

RESUMO

BACKGROUND/AIM: Hypoxia offers resistance to therapy in human solid tumors. The aim of the study was to investigate whether SN-38, the active metabolite of irinotecan, acts as a radiosensitizer through inhibition of hypoxia-inducible factor (HIF)-1α in the human colorectal cancer (CRC) cells. MATERIALS AND METHODS: HT29 and SW480 cells were cultured with SN-38 (0-4 µM) immediately after irradiation (0-8 Gy). HIF-1α expression was assessed using flow-cytometry and western blot analysis. Cell proliferation was evaluated by the calcein assay. Apoptosis and cell cycle were determined by flow-cytometry. RESULTS: Radiation up-regulated HIF-1α, and SN-38 inhibited the radiation-induced HIF-1α. The combination of radiation and SN-38 inhibited cell proliferation more than radiation alone; treatment with SN-38 after radiation exposure did not increase the number of apoptotic cells, whereas, it enhanced the S and G2/M cell-cycle arrest and decreased the population of cells in G1 Conclusion: SN-38 inhibits the radiation-induced up-regulation of HIF-1α and acts as a radiosensitizer by inducing cell-cycle arrest in CRC cells.


Assuntos
Camptotecina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Radiossensibilizantes/farmacologia , Regulação para Cima/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Células HT29 , Humanos , Irinotecano , Fase S/efeitos dos fármacos , Fase S/efeitos da radiação , Regulação para Cima/efeitos da radiação
20.
J Radiat Res ; 59(4): 411-429, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29800458

RESUMO

Gene expression analysis was carried out in Jurkat cells in order to identify candidate genes showing significant gene expression alterations allowing robust discrimination of the Auger emitter 123I, incorporated into the DNA as 123I-iododeoxyuridine (123IUdR), from α- and γ-radiation. The γ-H2AX foci assay was used to determine equi-effect doses or activity, and gene expression analysis was carried out at similar levels of foci induction. Comparative gene expression analysis was performed employing whole human genome DNA microarrays. Candidate genes had to show significant expression changes and no altered gene regulation or opposite regulation after exposure to the radiation quality to be compared. The gene expression of all candidate genes was validated by quantitative real-time PCR. The functional categorization of significantly deregulated genes revealed that chromatin organization and apoptosis were generally affected. After exposure to 123IUdR, α-particles and γ-rays, at equi-effect doses/activity, 155, 316 and 982 genes were exclusively regulated, respectively. Applying the stringent requirements for candidate genes, four (PPP1R14C, TNFAIP8L1, DNAJC1 and PRTFDC1), one (KLF10) and one (TNFAIP8L1) gene(s) were identified, respectively allowing reliable discrimination between γ- and 123IUdR exposure, γ- and α-radiation, and α- and 123IUdR exposure, respectively. The Auger emitter 123I induced specific gene expression patterns in Jurkat cells when compared with γ- and α-irradiation, suggesting a unique cellular response after 123IUdR exposure. Gene expression analysis might be an effective tool for identifying biomarkers for discriminating different radiation qualities and, furthermore, might help to explain the varying biological effectiveness at the mechanistic level.


Assuntos
Partículas alfa , Biomarcadores/metabolismo , Raios gama , Perfilação da Expressão Gênica , Idoxuridina/farmacologia , DNA/metabolismo , Dano ao DNA , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Estudos de Associação Genética , Histonas/metabolismo , Humanos , Células Jurkat , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
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