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PURPOSE: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options. METHODS: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice. RESULTS: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line. CONCLUSION: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.
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Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Proteínas da Gravidez/metabolismo , Fatores Supressores Imunológicos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Quinase 1 do Ponto de Checagem/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas da Gravidez/biossíntese , Proteínas da Gravidez/genética , Transdução de Sinais , Fatores Supressores Imunológicos/biossíntese , Fatores Supressores Imunológicos/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais CultivadasRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been characterized as an anti-cancer therapeutic agent with prominent cancer cell selectivity over normal cells. However, breast cancer cells are generally resistant to TRAIL, thus limiting its therapeutic potential. In this study, we found that BIX-01294, a selective inhibitor of euchromatin histone methyltransferase 2/G9a, is a strong TRAIL sensitizer in breast cancer cells. The combination of BIX-01294 and TRAIL decreased cell viability and led to an increase in the annexin V/propidium iodide-positive cell population, DNA fragmentation, and caspase activation. BIX-01294 markedly increased death receptor 5 (DR5) expression, while silencing of DR5 using small interfering RNAs abolished the TRAIL-sensitizing effect of BIX-01294. Specifically, BIX-01294 induced C/EBP homologous protein (CHOP)-mediated DR5 gene transcriptional activation and DR5 promoter activation was induced by upregulation of the protein kinase R-like endoplasmic reticulum kinase-mediated activating transcription factor 4 (ATF4). Moreover, inhibition of reactive oxygen species by N-acetyl-L-cysteine efficiently blocked BIX-01294-induced DR5 upregulation by inhibiting ATF4/CHOP expression, leading to diminished sensitization to TRAIL. These findings suggest that BIX-01294 sensitizes breast cancer cells to TRAIL by upregulating ATF4/CHOP-dependent DR5 expression with a reactive oxygen species-dependent manner. Furthermore, combination treatment with BIX-01294 and TRAIL suppressed tumor growth and induced apoptosis in vivo. In conclusion, we found that epigenetic regulation can contribute to the development of resistance to cancer therapeutics such as TRAIL, and further studies of unfolded protein responses and the associated epigenetic regulatory mechanisms may lead to the discovery of new molecular targets for effective cancer therapy.
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Fator 4 Ativador da Transcrição/metabolismo , Neoplasias da Mama/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Espécies Reativas de Oxigênio/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Azepinas/farmacologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Xenoenxertos , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Modelos Biológicos , Quinazolinas/farmacologia , Fator de Transcrição CHOP/metabolismoRESUMO
STUDY QUESTION: Does interleukin-32 (IL-32) play a role in the pathogenesis of endometriosis? SUMMARY ANSWER: IL-32 might be involved in the pathogenesis of endometriosis through increased viability, proliferation and invasion of endometrial cells. WHAT IS KNOWN ALREADY: Endometriosis is characterized as a chronic inflammatory disease and several proinflammatory cytokines are suggested to be involved in its pathogenesis and pathophysiology. IL-32, recognized as a new proinflammatory cytokine and a strong inducer of other proinflammatory cytokines, has been shown to serve as a key modulator in several chronic inflammatory diseases. STUDY DESIGN, SIZE, DURATION: This study included comparison of IL-32 levels in the peritoneal fluids between women with and without endometriosis, in-vitro experiments using Ishikawa cells and endometrial stromal cells (ESCs), and experiments on IL-32 transgenic mice and wild-type mice with induced endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: IL-32 levels in the peritoneal fluids were measured using enzyme-linked immunosorbent assays. Cell viability, expression of proliferating cell nuclear antigen (PCNA), and cellular invasiveness were analyzed following in-vitro treatment of Ishikawa cells and ESCs with recombinant IL-32 alpha (α) and gamma (γ). Ectopic endometriotic lesions were compared between IL-32 transgenic mice and wild-type mice after autologous endometrial transplantation with immunohistochemistry for Ki-67 antigen and PCNA. MAIN RESULTS AND THE ROLE OF CHANCE: The peritoneal fluid concentration of IL-32 was significantly higher in patients with advanced stage endometriosis compared with the controls. In-vitro treatment with IL-32 α and γ caused significant increases in cellular viability, PCNA expression, and invasiveness in Ishikawa cells and ESCs. The IL-32 transgenic mice had a significantly larger size of the ectopic endometrial lesions with higher expression of Ki-67 antigen and PCNA compared with wild-type mice. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: It is still unclear whether IL-32 is a main regulator, or one of several downstream proinflammatory cytokines, causing establishment and/or progression of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Further investigation on IL-32 signaling pathways may contribute to development a more effective treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant number: HI16C1682). None of the authors has anything to disclose.
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Líquido Ascítico/metabolismo , Endometriose/metabolismo , Endométrio/metabolismo , Interleucinas/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Inflamação/metabolismo , Interleucinas/farmacologia , Camundongos , Camundongos Transgênicos , Células Estromais/metabolismoRESUMO
OBJECTIVES: To validate a method for performing myocardial segmentation based on coronary anatomy using coronary CT angiography (CCTA). METHODS: Coronary artery-based myocardial segmentation (CAMS) was developed for use with CCTA. To validate and compare this method with the conventional American Heart Association (AHA) classification, a single coronary occlusion model was prepared and validated using six pigs. The unstained occluded coronary territories of the specimens and corresponding arterial territories from CAMS and AHA segmentations were compared using slice-by-slice matching and 100 virtual myocardial columns. RESULTS: CAMS more precisely predicted ischaemic area than the AHA method, as indicated by 95% versus 76% (p < 0.001) of the percentage of matched columns (defined as percentage of matched columns of segmentation method divided by number of unstained columns in the specimen). According to the subgroup analyses, CAMS demonstrated a higher percentage of matched columns than the AHA method in the left anterior descending artery (100% vs. 77%; p < 0.001) and mid- (99% vs. 83%; p = 0.046) and apical-level territories of the left ventricle (90% vs. 52%; p = 0.011). CONCLUSIONS: CAMS is a feasible method for identifying the corresponding myocardial territories of the coronary arteries using CCTA. KEY POINTS: ⢠CAMS is a feasible method for identifying corresponding coronary territory using CTA ⢠CAMS is more accurate in predicting coronary territory than the AHA method ⢠The AHA method may underestimate the ischaemic territory of LAD stenosis.
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Angiografia por Tomografia Computadorizada , Angiografia Coronária/métodos , Doença da Artéria Coronariana/diagnóstico por imagem , Vasos Coronários/diagnóstico por imagem , Coração/diagnóstico por imagem , Miocárdio/patologia , Animais , Modelos Animais de Doenças , SuínosRESUMO
Introduction: Humanized mouse models to recapitulate human biological systems still have limitations, such as the onset of lethal graft-versus-host disease (GvHD), a variable success rate, and the low accessibility of total body irradiation (TBI). Recently, mice modified with the CD47-SIRPA axis have been studied to improve humanized mouse models. However, such trials have been rarely applied in NOD mice. In this study, we created a novel mouse strain, NOD-CD47nullRag2nullIL-2rγnull (RTKO) mice, and applied it to generate humanized mice. Methods: Four-week-old female NOD-Rag2nullIL-2rγnull (RID) and RTKO mice pre-conditioned with TBI or busulfan (BSF) injection were used for generating human CD34+ hematopoietic stem cell (HSC) engrafted humanized mice. Clinical signs were observed twice a week, and body weight was measured once a week. Flow cytometry for human leukocyte antigens was performed at intervals of four weeks or two weeks, and mice were sacrificed at 48 weeks after HSC injection. Results: For a long period from 16 to 40 weeks post transplantation, the percentage of hCD45 was mostly maintained above 25% in all groups, and it was sustained the longest and highest in the RTKO BSF group. Reconstruction of human leukocytes, including hCD3, was also most prominent in the RTKO BSF group. Only two mice died before 40 weeks post transplantation in all groups, and there were no life-threatening GvHD lesions except in the dead mice. The occurrence of GvHD has been identified as mainly due to human T cells infiltrating tissues and their related cytokines. Discussion: Humanized mouse models under all conditions applied in this study are considered suitable models for long-term experiments based on the improvement of human leukocytes reconstruction and the stable animal health. Especially, RTKO mice pretreated with BSF are expected to be a valuable platform not only for generating humanized mice but also for various immune research fields.
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Bussulfano , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Camundongos Endogâmicos NOD , Camundongos Knockout , Condicionamento Pré-Transplante , Animais , Bussulfano/farmacologia , Humanos , Camundongos , Transplante de Células-Tronco Hematopoéticas/métodos , Condicionamento Pré-Transplante/métodos , Células-Tronco Hematopoéticas/metabolismo , Feminino , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/deficiência , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/imunologia , Modelos Animais de Doenças , Irradiação Corporal TotalRESUMO
BACKGROUND: Schwann cells (SCs) secrete neurotrophic factors and provide structural support and guidance during axonal regeneration. However, nearby nerves may be damaged to obtain primary SCs, and there is a lack of nervous tissue donors. We investigated the potential of Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) in differentiating into Schwann cell-like cells (WJ-SCLCs) as an alternative to SCs. We also examined whether implantation of WJ-SCLCs-laden acellular nerve grafts (ANGs) are effective in inducing functional recovery and nerve regeneration in an animal model of peripheral nerve injury. METHODS: The differentiation of WJ-MSCs into WJ-SCLCs was determined by analyzing SC-specific markers. The secretion of neurotrophic factors was assessed by the Neuro Discovery antibody array. Neurite outgrowth and myelination of axons were found in a co-culture system involving motor neuron cell lines. The effects of ANGs on repairing sciatic nerves were evaluated using video gait angle test, isometric tetanic force analysis, and toluidine blue staining. RESULTS: Compared with undifferentiated WJ-MSCs, WJ-SCLCs showed higher expression levels of SC-specific markers such as S100ß, GFAP, KROX20, and NGFR. WJ-SCLCs also showed higher secreted amounts of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and granulocyte-colony stimulating factor than did WJ-MSCs. WJ-SCLCs effectively promoted the outgrowth and myelination of neurites in motor neuron cells, and WJ-SCLCs laden ANGs significantly facilitated peripheral nerve regeneration in an animal model of sciatic nerve injury. CONCLUSION: WJ-MSCs were readily differentiated into WJ-SCLCs, which effectively promoted the regeneration of peripheral nerves. Transplantation of WJ-SCLCs with ANGs might be useful for assisting peripheral nerve regeneration.
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Células-Tronco Mesenquimais , Geleia de Wharton , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Regeneração Nervosa , Células de Schwann , Nervo IsquiáticoRESUMO
Evidence is growing that phthalate esters play an important role in the pathogenesis of estrogen-dependent gynecologic diseases, especially uterine fibroids. We aimed to investigate whether in vitro treatment with di-(2-ethylhexyl)-phthalate (DEHP) affects angiogenesis, proliferation, and apoptosis in uterine fibroids. To ascertain this, we evaluated vascular endothelial growth factor (VEGF) expression and AKT/ERT phosphorylation and compared the fibroid volume between nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice fed with and without DEHP. VEGF expression was measured using enzyme-linked immunosorbent assay, and AKT/ERK phosphorylation was analyzed by western blot analysis in human myometrial and fibroid cells. The volume of the fibroid tissues implanted to NOD/SCID mice was measured, and the expression of collagen type I protein, Ki-67, proliferating cell nuclear antigen, and B cell lymphoma 2 were analyzed using immunohistochemistry. We could see significant increases in VEGF expression and AKT phosphorylation in human myometrial and fibroid cells treated with DEHP. The volume of the fibroid tissues was significantly increased in NOD/SCID mice fed with DEHP, which was accompanied by increased expression of collagen type I and AKT phosphorylation. Taken together, these results suggest that exposure to phthalate esters may influence uterine fibroid pathogenesis by increasing VEGF and collagen expression and upregulating AKT phosphorylation.
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Dietilexilftalato/toxicidade , Ésteres/toxicidade , Leiomioma/patologia , Miométrio/efeitos dos fármacos , Neoplasias Uterinas/patologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Leiomioma/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Miométrio/metabolismo , Miométrio/patologia , Neovascularização Patológica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias Uterinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases.Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.
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Tecido Adiposo Marrom/metabolismo , Autofagia/fisiologia , Inflamassomos/metabolismo , Mitofagia/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Adipócitos/metabolismo , Animais , Metabolismo Energético/fisiologia , Camundongos Knockout , Mitocôndrias/metabolismo , Mitofagia/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
In the event of a mass casualty radiation scenario, biodosimetry has the potential to quantify individual exposures for triaging and providing dose-appropriate medical intervention. Structural maintenance of chromosomes 1 (SMC1) is phosphorylated in response to ionizing radiation. The goal of this study was to develop a new biodosimetry method using SMC1 phosphorylation as a measure of exposure to radiation. In the initial experiments, two normal human cell lines (WI-38VA-13 and HaCaT) and four lymphoblastoid cell lines were irradiated, and the levels of SMC1 phosphorylation at Ser-360 and Ser-957 were assessed using Western blotting. Subsequently, similar experiments were performed using peripheral blood mononuclear cells (PBMCs) obtained from 20 healthy adults. Phosphorylation of SMC1 at Ser-957 and Ser-360 was increased by exposure in a dose-dependent manner, peaked at 1-3 h postirradiation and then decreased gradually. Ser-360 was identified as a new phosphorylation site and was more sensitive to radiation than Ser-957, especially at doses below 1 Gy. Our results demonstrate a robust ex vivo response of phospho-SMC1-(Ser-360) to ionizing radiation in human PBMCs. Detection of phosphorylation at Ser-360 in SMC1 could be used as a marker of radiation exposure. Our findings suggest that it is feasible to measure blood cell-based changes in the phosphorylation level of a protein as an ex vivo radiation exposure detection method, even after low-dose exposure.
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Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Serina/metabolismo , Linhagem Celular , Cromatina/metabolismo , Relação Dose-Resposta à Radiação , Humanos , Fosforilação/efeitos da radiação , Fatores de TempoRESUMO
The liver has marked regenerative capabilities, and numerous signaling pathways are involved in liver regeneration. The transforming growth factor-ß (TGF-ß)/Smad pathway, which is also involved in liver regeneration, regulates numerous biological processes. Krüppel-like factor 10 (KLF10) has been reported to activate the TGF-ß/Smad signaling pathway; however, the exact functions of KLF10 under various pathophysiological conditions remain unclear. In the present study, the role of KLF10 in liver regeneration following partial hepatectomy (PH) was investigated using KLF10-knockout (KO) mice. KLF10-KO mice exhibited lower liver/body weight ratios and 5-bromo-2-deoxy-uridine labeling indices compared with wild-type (WT) mice, and significant differences (P=0.028) were obtained at 72 h after PH. To understand the causes of the gross and histopathological findings, the expression levels of the components of the TGF-ß/Smad pathway were examined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The mRNA and protein levels of Smad3, p15, TGF-ß1 and TGF-ß receptor 1 were significantly increased, while those of cMyc and cyclin D1 (proliferation-associated genes) were significantly lower in the liver tissues of the KLF10-KO mice compared with those of the WT mice at 72 h post-PH. These results indicated that KLF10-KO may exhibit antiproliferative effects on liver regeneration following PH, through strengthening the TGF-ß/Smad signaling pathway in a delayed manner.
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AIM: We investigated the therapeutic effects of a mitogen-activated protein (MEK) inhibitor, selumetinib, in a hepatic melanoma metastasis model and studied its possible mechanism of action. MATERIALS AND METHODS: Melanoma cell lines were exposed to selumetinib under different experimental conditions. We established a mouse model of liver metastasis and treated mice orally with vehicle or selumetinib and then evaluated metastasis progress. RESULTS: Growth inhibition was observed in melanoma cells as a consequence of G1-phase cell-cycle arrest and the subsequent induction of apoptosis in a dose- and time-dependent manner. Mice with established liver metastases that were treated with selumetinib exhibited significantly less tumor progression than vehicle-treated mice. c-Myc expression in metastasized liver tissues were suppressed by selumetinib. Moreover, oral treatment with selumetinib modulated expression of epithelial-to-mesenchymal transition- and metastasis-related genes, including integrin alpha-5 (ITGA5), jagged 1 (JAG1), zinc finger E-box-binding homeobox 1 (ZEB1), NOTCH, and serpin peptidase inhibitor clade E (SERPINE1). CONCLUSION: We established a mouse model of hepatic metastasis using a human melanoma cell line, such models are essential in elucidating the therapeutic effects of anti-metastatic drugs. Our data suggest the possibility that selumetinib presents a new strategy to treat liver metastasis in patients with melanoma by suppressing epithelial-to-mesenchymal transition-related genes.
Assuntos
Benzimidazóis/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Hepáticas/prevenção & controle , Melanoma/tratamento farmacológico , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzimidazóis/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Integrina alfa5/genética , Integrina alfa5/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Masculino , Melanoma/genética , Melanoma/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Radiation-induced fibrosis (RIF) is one of the most common late complications of radiation therapy. We found that α-lipoic acid (α-LA) effectively prevents RIF. In RIF a mouse model, leg contracture assay was used to test the in vivo efficacy of α-LA. α-LA suppressed the expression of pro-fibrotic genes after irradiation, both in vivo and in vitro, and inhibited the up-regulation of TGF-ß1-mediated p300/CBP activity. Thus, α-LA prevents radiation-induced fibrosis (RIF) by inhibiting the transcriptional activity of NF-κB through inhibition of histone acetyltransferase activity. α-LA is a new therapeutic methods that can be used in the prevention-treatment of RIF.
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Fibrose/prevenção & controle , Lesões por Radiação/prevenção & controle , Protetores contra Radiação/farmacologia , Ácido Tióctico/farmacologia , Animais , Antioxidantes/farmacologia , Fibrose/etiologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Streptococcus pneumoniae is a major human pathogen that is involved in community-acquired pneumonia. Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine that activates immune responses against infection, invasion, injury, or inflammation. To study the role of TNF-α during S. pneumoniae infection, a murine pneumococcal pneumonia model was used. We intranasally infected C57BL/6J wild-type (WT) and TNF-α knockout (KO) mice with S. pneumoniae D39 serotype 2. In TNF-α KO mice, continuous and distinct loss of body weight, and low survival rates were observed. Bacterial counts in the lungs and blood of TNF-α KO mice were significantly higher than those in WT mice. Histopathological lesions in the spleen of TNF-α KO mice were more severe than those in WT mice. In TNF-α KO mice, severe depletion of white pulp was observed and the number of apoptotic cells was significantly increased. Interferon-gamma (IFN-γ), IL-12p70 and IL-10 levels in serum were significantly increased in TNF-α KO mice. TNF-α is clearly involved in the regulation of S. pneumoniae infections. Early death and low survival rates of TNF-α KO mice were likely caused by a combination of impaired bacterial clearance and damage to the spleen. Our findings suggest that TNF-α plays a critical role in protecting the host from systemic S. pneumoniae infection.
RESUMO
Liver cancer is the third most common cancer, and the incidence as well as the mortality rate of liver cancer are on the increase. There are many signaling pathways that are involved in hepatic tumorigenesis. One of these pathways, the transforming growth factor-ß (TGF-ß)/Smad pathway with KLF10, has been reported to suppress cellular proliferation in most cases. However, the actual functions of KLF10 in various pathophysiological conditions are still fragmentary and unclear. In the present study, the practical role of KLF10 in DEN-induced hepatic carcinogenesis, was elucidated using KLF10 null mice. In the necropsy and histopathological analysis, KLF10 KO mice exhibited lower tumor incidence and PCNA labeling indices than these values in the wild-type mice. Additional analyses revealed that the mRNA and protein levels of Smad3, TGF-ß1, TGF-ß RI and p15 were increased in the tumor tissues of the KLF10 KO mice, while those of cMyc and cyclin D1 were downregulated. The level of phospho-Smad3 was also significantly higher in the tumor tissues of the KLF10 KO mice. All together, the KLF10 KO condition may reinforce the TGF-ßSmad signaling pathway and confer tumor-suppressor effects against chemically induced liver tumorigenesis.
Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Hepáticas/genética , Proteínas Supressoras de Tumor/genética , Animais , Ciclina D1/genética , Incidência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/genéticaRESUMO
CONTEXT: Although phthalates were shown to have several negative effects on reproductive function in animals, its role in the pathogenesis of endometriosis remains to be elucidated. OBJECTIVE: We aimed to investigate the in vitro and in vivo effects of di-(2-ethylhexyl)-phthalate (DEHP) and to compare the urinary levels of several phthalate metabolites between women with and without endometriosis. DESIGN: For experimental studies, we used endometrial cell culture and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse models. We also performed a prospective case-control study for human sample analyses. SETTING: The study was conducted at an academic center. MAIN OUTCOME MEASURES: The activities of matrix metalloproteinase (MMP)-2 and 9, cellular invasiveness, phosphorylation of extracellular signal-regulated kinase (Erk), and expression of p21-activated kinase 4 were analyzed in endometrial cells treated with DEHP. The implant size was compared between NOD/SCID mice fed with and without DEHP. Urinary concentrations of several phthalate metabolites were compared between women with and without endometriosis. RESULTS: In vitro treatment of endometrial cells with DEHP led to significant increases of MMP-2 and 9 activities, cellular invasiveness, Erk phosphorylation, and p21-activated kinase 4 expression. The size of the endometrial implant was significantly larger in the NOD/SCID mice fed with DEHP compared with those fed with vehicle. The urinary concentration of mono (2-ethyl-5-hydroxyhexyl) phthalate, mono (2-ethyl-5-oxohexyl) phthalate, and mono (2-ethyl-5-carboxyphentyl) phthalate were significantly higher in women with endometriosis compared with controls. CONCLUSION: These findings strongly suggest that exposure to phthalate may lead to establishment of endometriosis by enhancing invasive and proliferative activities of endometrial cells.
Assuntos
Dietilexilftalato/toxicidade , Endometriose/induzido quimicamente , Plastificantes/toxicidade , Animais , Estudos de Casos e Controles , Células Cultivadas , Endometriose/epidemiologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Ácidos Ftálicos/urina , Estudos Prospectivos , Células Estromais/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae are important pathogens that cause respiratory infections in laboratory rodents. In this study, we used species-specific triplex PCR analysis to directly detect three common bacterial pathogens associated with respiratory diseases. Specific targets were amplified with conventional PCR using the tyrB gene from K. pneumoniae, gyrB gene from C. kutscheri, and ply gene from S. pneumoniae. Our primers were tested against purified DNA from another eleven murine bacteria to determine primer specificity. Under optimal PCR conditions, the triplex assay simultaneously yielded a 931 bp product from K. pneumoniae, a 540 bp product from C. kutscheri, and a 354 bp product from S. pneumoniae. The triplex assay detection thresholds for pure cultures were 10 pg for K. pneumoniae and S. pneumoniae, and 100 pg for C. kutscheri. All three bacteria were successfully identified in the trachea and lung of experimentally infected mice at the same time. Our triplex PCR method can be used as a useful method for detecting pathogenic bacterial infections in laboratory rodents.
Assuntos
Animais de Laboratório/microbiologia , Corynebacterium/isolamento & purificação , Klebsiella pneumoniae/isolamento & purificação , Camundongos Endogâmicos BALB C/microbiologia , Reação em Cadeia da Polimerase/métodos , Infecções Respiratórias/microbiologia , Infecções Respiratórias/veterinária , Doenças dos Roedores/microbiologia , Streptococcus pneumoniae/isolamento & purificação , Animais , Corynebacterium/genética , DNA Bacteriano/isolamento & purificação , Klebsiella pneumoniae/genética , Pulmão/microbiologia , Camundongos , Streptococcus pneumoniae/genética , Traqueia/microbiologiaRESUMO
Gastric cancer is one of the major public health problems. Despite new chemotherapeutic treatments, the prognosis of gastric cancer remains poor. 5-Fluorouracil (5-FU) is used as a standard chemotherapy drug in gastric cancer. However, 5-FU resistance develops frequently and is a main cause of chemotherapy failure in human gastric cancer. Overexpression of cyclin D1 is related to rapid cell growth, a poor prognosis and increased chemoresistance in several types of cancers. In this study, we investigated whether treatment of gastric cancer cells with shRNA targeting cyclin D1 (ShCCND1) or 5-FU, alone or in combination, influences the activation of phosphorylated AKT (pAKT) and pNFκB, which are markers that are increased in 5-FU chemoresistance. We also investigated the effect of combined treatment with ShCCND1 and 5-FU on cell growth and chemosensitivity to 5-FU in the gastric cancer cell line AGS. The data showed that ShCCND1-mediated cyclin D1 downregulation in AGS cells significantly inhibited cell proliferation, cell mobility and clonogenicity. In addition, combined treatment with ShCCND1 and 5-FU significantly decreased the survival rate of AGS cells, compared to single-treatment with either agent. These results demonstrated that ShCCND1 increases 5-FU chemosensitivity, a conclusion that is also supported by the concomitant reduction in expression of pAKT and pNFκB, increase of G1 arrest and induction of apoptosis. Taken together, these data provide further evidence that therapeutic strategies targeting cyclin D1 may have the dual advantage of suppressing the growth of cancer cells, while enhancing their chemosensitivity.
Assuntos
Ciclina D1/genética , Fluoruracila/farmacologia , RNA Interferente Pequeno/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Ciclina D1/biossíntese , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Humanos , Lentivirus/genética , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNARESUMO
Deregulated Wnt signaling pathway is implicated in many hereditary diseases and tumorigenesis including colorectal cancer, hepatocellular carcinoma and gastric cancer. In this study, to assess the relationship between chemically induced gastric tumor and canonical Wnt signaling pathway in genetically intact mice, histopathological and quantitative mRNA analyses were performed in C57BL/6J mice given drinking water containing N-methyl- N-nitrosurea (MNU). 60.5% of gastric adenoma and 27.9% of adenocarcinoma were observed 48 weeks after first administration. Also, in immunohistochemical analysis, aberrant expressions of phospho-GSK-3ß, ß-catenin, cyclin D1, c-Myc, osteopontin and COX-2 were found. In double immunofluorescent-antibody stains, ß-catenin accumulation was colocalized with other proteins. mRNA levels of cyclin D1, c-myc and COX-2 were relatively higher in adenocarcinoma. Altogether, canonical Wnt pathway was highly involved in MNU induced gastric neoplasia of C57BL/6J mice, and it could be a considerably suitable system for the study to examine the linkage between gastric tumorigenesis and the canonical Wnt pathway.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metilnitrosoureia/toxicidade , Neoplasias Gástricas/induzido quimicamente , Via de Sinalização Wnt/fisiologia , Animais , Imuno-Histoquímica , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/metabolismoRESUMO
Interleukin (IL)-10 is an anti-inflammatory cytokine that modulates sepsis by decreasing pro-inflammatory cytokine production and chemokine expression. In this study, IL-10-deficient and wild-type (WT) mice were infected with Corynebacterium kutscheri to determine if the absence of IL-10 altered the protective immunity and pathogenesis. After infection, IL-10 knockout (KO) mice had a higher survival rate than WT mice. The decrease of body weight and the increased weight of organs such as liver and spleen were greater in WT mice. Bacterial counts were significantly increased after inoculation in WT mice over those in IL-10 KO mice. WT mice had more granulomatous inflammation and coagulative necrosis in the liver and spleen, lymphocyte depletion in lymphoid follicles, and apoptosis of immune cells in the spleen. WT mice had significantly higher plasma concentrations of aspartate aminotransferase and alanine aminotransferase. Furthermore, more upregulation of tumor necrosis factor-α and IL-4 in the plasma, macrophage inflammatory protein-2, keratinocyte-derived chemokine, inducible nitric oxide synthase, and interferon-inducible protein 10 mRNA in the spleen were observed in WT mice after inoculation. These results suggest that the lack of IL-10 contributes to an increase in the systemic clearance of C. kutscheri, and that IL-10 plays a detrimental role in controlling systemic C. kutscheri infection.
Assuntos
Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Corynebacterium/fisiologia , Interleucina-10/imunologia , Animais , Corynebacterium/imunologia , Infecções por Corynebacterium/genética , Humanos , Fígado/imunologia , Masculino , Camundongos , Camundongos Knockout , Baço/imunologiaRESUMO
The accurate and economical diagnosis of pathogenic bacteria is necessary for the microbiological control of laboratory animals. In this study, we developed a triplex PCR method for the direct detection of three common gastroenteric bacteria, Pseudomonas aeruginosa, Helicobacter hepaticus, and Salmonella typhimurium. Targets were specifically amplified by conventional PCR assay using a genomic fragment from P. aeruginosa, 16S ribosomal RNA from H. hepaticus, and the invA gene from S. typhimurium. To investigate the specificity of our primers, they were tested against purified DNA from many other bacterial species. There were no amplification products from other bacteria. Under optimized conditions, the triplex assay simultaneously yielded a 726-bp product from P. aeruginosa, a 417-bp product from H. hepaticus, and a 246-bp product from S. typhimurium. The detection limits of this assay in pure culture were 10 pg for P. aeruginosa, and 0.1 pg for H. hepaticus and S. typhimurium. All three bacteria were successfully detected in the liver, cecum, and feces of experimentally infected mice. This method is a useful and convenient assay that allows the simultaneous identification of bacterial pathogens in mice. Our triplex method will be used to improve quality control in the detection of pathogenic bacterial infections in laboratory animal facilities.