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1.
Zhonghua Yi Xue Za Zhi ; 99(34): 2701-2705, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31505723

RESUMO

Objective: To established Apc(loxp/loxp)+Kras(LSL-G12D/-)+Smad4(loxp/loxp) transgenic mouse model that mimick the occurrence and development of human sporadic colorectal cancer(CRC) and its liver metastasis. Methods: C57BL/6-Apc(tm1Tyj)/J(Apc(loxP)), B6.129S4-Kras(tm4Tyj)/J(Kras(LSL-G12D)), 129S6-Smad4(tm2.1Cxd)/J(Smad4(loxP)) and C57BL/6J mice were crossed, and genotype with Apc(loxP/loxP)+Kras(LSL-G12D/-)and Apc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP)were generated. Genotypes of the mice were identified by PCR and real-time quantitative PCR. The mice were divided into Apc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP) group (n=20) and Apc(loxP/loxP)+Kras(LSL-G12D/-)group(n=24). Lentivirus expressing Cre enzyme and IRES-luciferase were injected into the submucosa of colon or rectum of the transgenic mice under colonoscopy. Intraabdominal injection of D-luciferase into mice every 4 weeks, imaging with small animal in vivo imaging system(IVIS). The tumor size, tumorigenesis rate and metastasis ratio were analyzed. At the end of the 20th week, the colorectal lesions and metastatic tissues of mice were stained with hematoxylin-eosin(HE) and the pathological changes were observed under microscope. Results: Apc(loxp/loxp)+Kras(LSL-G12D/-)+Smad4(loxp/loxp) and Apc(loxp/loxp)+Kras(LSL-G12D/-)transgenic mice were successfully bred. The colorectal stem cells of the transgenic mouse mutated leading tumor lesion and liver metastatic under the induction of Lentivirus(Cre-IRES-luciferase). The primary and metastatic foci of colorectal carcinoma and liver metastasis in mice were proved to be adenocarcinoma and liver metastatic carcinoma by histopathological examination. The primary tumor size inApc(loxP/loxP)+Kras(LSL-G12D/-)+Smad4(loxP/loxP) group and Apc(loxP/loxP)+Kras(LSL-G12D/-)group was(3.52±0.26) and(3.45±0.20)mm, respectively,without significant difference(t=0.872, P=0.388).The tumorigenesis rate was 70.0% and 50.0% respectively, and there was no significant difference between the two groups(χ(2)=0.440, P=0.507). The metastasis rate of two groups were 58.3% and 8.3%respectively(Fisher's exact test, P=0.027). Conclusions: In this study, the colorectal carcinogenesis and its spontaneously metastasis to the liver of CRC were induced by Lentivirus(Cre-IRES-luciferase) in our established transgenic mice,which successfully simulated the occurrence and development of human sporadic CRC and its liver metastasis.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Neoplasias Pancreáticas , Animais , Progressão da Doença , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)
2.
Anticancer Res ; 39(8): 4061-4064, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366488

RESUMO

BACKGROUND/AIM: Circulating tumor cells (CTCs) may have an important role in metastasis. CTC clusters, which contain fibroblasts, indicate poor prognosis. In the present study, we used our malignant lymphoma metastatic mouse model to compare the effect of a choline-deficient-diet (CDD) and the control diet (CD) on fibroblasts in CTCs. MATERIALS AND METHODS: We compared the number and morphology of CTCs in CDD and CD mice using color-coded imaging with fluorescent proteins. Malignant lymphoma EL4 cells expressing RFP were injected in the spleen of transgenic C57B/6-GFP mice, which were fed a CDD or CD. Two weeks later, we harvested and observed the number of CTCs and fibroblast-like cells both in heart blood and portal blood. Imaging of CTC morphology was performed with smeared glass slides and in culture. RESULTS AND CONCLUSION: There was no significant difference in the number of CTCs between CDD and CD mice. The number of fibroblast-like cells in the CTC microenvironment in CD mouse portal blood was significantly larger than in CDD mouse portal blood. These differences may be caused by deficiency in choline that leads to less metastasis in choline-deficient-diet-induced fatty liver.


Assuntos
Colina/metabolismo , Linfoma/sangue , Células Neoplásicas Circulantes/metabolismo , Células Estromais/metabolismo , Animais , Linhagem Celular Tumoral , Deficiência de Colina/sangue , Deficiência de Colina/genética , Deficiência de Colina/patologia , Dieta/efeitos adversos , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Fluorescência Verde/química , Humanos , Proteínas Luminescentes/química , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , Células Estromais/patologia , Microambiente Tumoral/genética
3.
J Agric Food Chem ; 67(35): 9782-9788, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31390859

RESUMO

Sulforaphane, a potent antioxidant compound, is unstable at ambient temperature, whereas its precursor glucoraphanin is stable and metabolized to sulforaphane. Thus, we hypothesized that glucoraphanin-rich diet could effectively induce antioxidant enzyme activities and investigated the protective effects of long-term intake of a glucoraphanin-enriched kale (GEK) diet on skin aging in senescence-accelerated mouse prone 1 (SAMP1) mice. The senescence grading score was significantly lower after treatment with GEK for 39 weeks than that of the control mice. GEK also suppressed the thinning of the dorsal skin layer. Moreover, the GEK treatment enhanced the collagen production and increased the nuclear translocation of Nrf2 and HO-1 expression level in the skin tissue. TßRII and Smad3 expressions were clearly higher in the GEK-treated group than in the control group. Thus, GEK suppressed senescence in SAMP1 mice by enhancing the antioxidant activity and collagen production via the TßRII/Smad3 pathway, suggesting its practical applications for protection against skin aging.


Assuntos
Brassica/metabolismo , Glucosinolatos/metabolismo , Imidoésteres/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Extratos Vegetais/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Envelhecimento da Pele/fisiologia , Proteína Smad3/metabolismo , Animais , Antioxidantes/metabolismo , Brassica/química , Colágeno/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Transdução de Sinais , Envelhecimento da Pele/genética , Proteína Smad3/genética , Fatores de Tempo
4.
J Agric Food Chem ; 67(35): 9796-9804, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31393712

RESUMO

Overactivated microglia and persistent neuroinflammation hold an important role in the pathophysiology of neurodegenerative diseases. The extract of Lycoris chejuensis (CJ) and its active compound, 7-deoxy-trans-dihydronarciclasine (named E144), attenuated expressions of pro-inflammatory factors, including nitric oxide, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), and interleukin 6, secreted by lipopolysaccharide-activated BV-2 microglial cells, as measured by an enzyme-linked immunosorbent assay or western blotting. In contrast, CJ extract and E144 promoted the secretion of the anti-inflammatory cytokine, interleukin 10. Moreover, we found that E144 attenuated the expression of TNF-α and COX-2 in the cerebral cortex of lipopolysaccharide-treated mice and/or T2576 transgenic mice as well as reduced the reactive immune cells visualized by ionized calcium-binding adaptor molecule 1. Our results suggest the possibility of E144 to serve as a potential anti-neuroinflammatory agent by preventing excess production of pro-inflammatory factors.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/imunologia , Isoquinolinas/administração & dosagem , Lycoris/química , Extratos Vegetais/administração & dosagem , Doença de Alzheimer/genética , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Modelos Animais de Doenças , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Isoquinolinas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , Extratos Vegetais/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
5.
Nat Commun ; 10(1): 2919, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266962

RESUMO

Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specific phosphorylation of ERK in response to transgenic KRASG12V in mouse intestinal organoids, while transgenic BRAFV600E activates ERK in all cells. Quantitative network modelling from perturbation data reveals that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of ERK in the intestine. Furthermore, we find that oncogenic KRAS, together with ß-Catenin, favours expansion of crypt cells with high ERK activity. Our experiments highlight key differences between oncogenic BRAF and KRAS in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.


Assuntos
Neoplasias do Colo/enzimologia , Mucosa Intestinal/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Especificidade da Espécie
6.
Genome Biol ; 20(1): 140, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307522

RESUMO

BACKGROUND: Despite continual progress in the identification and characterization of trait- and disease-associated variants that disrupt transcription factor (TF)-DNA binding, little is known about the distribution of TF binding deactivating mutations (deMs) in enhancer sequences. Here, we focus on elucidating the mechanism underlying the different densities of deMs in human enhancers. RESULTS: We identify two classes of enhancers based on the density of nucleotides prone to deMs. Firstly, fragile enhancers with abundant deM nucleotides are associated with the immune system and regular cellular maintenance. Secondly, stable enhancers with only a few deM nucleotides are associated with the development and regulation of TFs and are evolutionarily conserved. These two classes of enhancers feature different regulatory programs: the binding sites of pioneer TFs of FOX family are specifically enriched in stable enhancers, while tissue-specific TFs are enriched in fragile enhancers. Moreover, stable enhancers are more tolerant of deMs due to their dominant employment of homotypic TF binding site (TFBS) clusters, as opposed to the larger-extent usage of heterotypic TFBS clusters in fragile enhancers. Notably, the sequence environment and chromatin context of the cognate motif, other than the motif itself, contribute more to the susceptibility to deMs of TF binding. CONCLUSIONS: This dichotomy of enhancer activity is conserved across different tissues, has a specific footprint in epigenetic profiles, and argues for a bimodal evolution of gene regulatory programs in vertebrates. Specifically encoded stable enhancers are evolutionarily conserved and associated with development, while differently encoded fragile enhancers are associated with the adaptation of species.


Assuntos
Adaptação Biológica , Elementos Facilitadores Genéticos , Evolução Molecular , Mutação , Animais , Genes Reporter , Células Hep G2 , Humanos , Camundongos Transgênicos
7.
Cancer Immunol Immunother ; 68(8): 1303-1315, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31278476

RESUMO

Our previous work has demonstrated the high efficiency of CD8+ natural killer T (NKT)-like cells in killing antigen-bearing dendritic cells. To evaluate their role in the tumor microenvironment, we performed in vitro and in vivo antitumor experiments to investigate whether CD8+NKT-like cells could kill Yac-1 and B16 cells like NK cells and kill EL4-OVA8 cells in an antigen-specific manner like cytotoxic T lymphocytes (CTLs). Unlike NK1.1-CTLs, CD8+NKT-like cells also exhibit the capability to kill myeloid-derived suppressor cells (MDSCs) in an antigen-specific manner, indicative of their potential role in clearing tumor antigen-bearing MDSCs to improve the antitumor microenvironment. In vitro blocking experiments showed that granzyme B inhibitor efficiently suppressed the cytotoxicity of CD8+NKT-like cells against tumor cells and MDSCs, while Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) inhibition failed to produce similar effects. Transcriptomic and phenotypic analyses of CD8+NKT-like cells, NK cells, and NK1.1-CTLs indicated that CD8+NKT-like cells expressed both T-cell activation markers and NK cell markers, thus bearing features of both the activated T cells and NK cells. Taken together, CD8+NKT-like cells could exert NK- and CTL-like antitumor effects through the elimination of both tumor cells and MDSCs in a granzyme B-dependent manner.


Assuntos
Células Matadoras Naturais/imunologia , Células Supressoras Mieloides/imunologia , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos CD8/metabolismo , Citotoxicidade Imunológica , Feminino , Granzimas/metabolismo , Humanos , Ativação Linfocitária , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Transcriptoma , Microambiente Tumoral
8.
Gene ; 714: 143996, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348980

RESUMO

The uniquely human α7-nAChR gene (CHRFAM7A) is evolved from the fusion of two partially duplicated genes, FAM7 and α7-nAChR gene (CHRNA7), and is inserted on same chromosome 15, 5' end of the CHRNA7 gene. Transcription of CHRFAM7A gene produces a 1256-bp open reading frame encoding dup-α7-nAChR, where a 27-aminoacid residues from FAM7 replaced the 146-aminoacid residues of the N-terminal extracellular ligand binding domain of α7-nAChR. In vitro, dup-α7-nAChR has been shown to form hetero-pentamer with α7-nAChR and dominant-negatively regulates the channel functions of α7-nAChR. However, the contribution of CHRFAM7A gene to the biology of α7-nAChR in the brain in vivo remains largely a matter of conjecture. CHRFAM7A transgenic mouse was created and differentially expressed proteins were profiled from the whole brain using iTRAQ-2D-LC-MS/MS proteomic technology. Proteins with a fold change of ≥1.2 or ≤0.83 and p < 0.05 were considered to be significant. Bioinformatics analysis showed that over-expression of the CHRFAM7A gene significantly modulated the proteins commonly involved in the signaling pathways of α7-nAChR-mediated neuropsychiatric disorders including Parkinson's disease, Alzheimer's disease, Huntington's disease, and alcoholism, suggesting that the CHRFAM7A gene contributes to the pathogenesis of neuropsychiatric disorders mostly likely through fine-tuning the functions of α7-nAChR in the brain.


Assuntos
Camundongos Transgênicos/genética , Receptor Nicotínico de Acetilcolina alfa7/genética , Animais , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Cromossomos Humanos Par 15/genética , Perfilação da Expressão Gênica/métodos , Genes Duplicados/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteômica/métodos , Transdução de Sinais/genética , Espectrometria de Massas em Tandem/métodos
9.
Ann Hematol ; 98(9): 2063-2072, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31312928

RESUMO

Rigosertib is a novel multi-kinase inhibitor, which has clinical activity towards leukemic progenitor cells of patients with high-risk myelodysplastic syndromes (MDS) after failure or progression on hypomethylating agents. Since the bone marrow microenvironment plays an important role in MDS pathogenesis, we investigated the impact of rigosertib on cellular compartments within the osteo-hematopoietic niche. Healthy C57BL/6J mice treated with rigosertib for 3 weeks showed a mild suppression of hematopoiesis (hemoglobin and red blood cells, both - 16%, p < 0.01; white blood cells, - 34%, p < 0.05; platelets, - 38%, p < 0.05), whereas there was no difference in the number of hematopoietic stem cells in the bone marrow. Trabecular bone mass of the spine was reduced by rigosertib (- 16%, p = 0.05). This was accompanied by a lower trabecular number and thickness (- 6% and - 10%, respectively, p < 0.05), partly explained by the increase in osteoclast number and surface (p < 0.01). Milder effects of rigosertib on bone mass were detected in an MDS mouse model system (NHD13). However, rigosertib did not further aggravate MDS-associated cytopenia in NHD13 mice. Finally, we tested the effects of rigosertib on human mesenchymal stromal cells (MSC) in vitro and demonstrated reduced cell viability at nanomolar concentrations. Deterioration of the hematopoietic supportive capacity of MDS-MSC after rigosertib pretreatment demonstrated by decreased number of colony-forming units, especially in the monocytic lineage, further supports the idea of disturbed crosstalk within the osteo-hematopoietic niche mediated by rigosertib. Thus, rigosertib exerts inhibitory effects on the stromal components of the osteo-hematopoietic niche which may explain the dissociation between anti-leukemic activity and the absence of hematological improvement.


Assuntos
Glicina/análogos & derivados , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas , Células-Tronco Mesenquimais , Síndromes Mielodisplásicas , Nicho de Células-Tronco/efeitos dos fármacos , Sulfonas/farmacologia , Animais , Glicina/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Transgênicos , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia
10.
Cancer Sci ; 110(9): 2748-2759, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301081

RESUMO

In hepatocarcinogenesis induced by diethylnitrosamine (DEN) in B6C3F1 mice, the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role. The livers of transgenic mice with a hepatocyte-specific human BRAFV600E mutation weighed 4.5 times more than that of normal mice and consisted entirely of hepatocytes, resembling DEN-induced preneoplastic hepatocytes. However, these transgenic mice spontaneously died 7 wk after birth, therefore this study aimed to clarify the causes of death. In the transgenic mice, the liver showed thrombopoietin (TPO) overexpression, which is associated with eventual megakaryocytosis and thrombocytosis, and activated platelets were deposited in hepatic sinusoids. TPO was also overexpressed in the DEN-induced hepatic tumors, and sinusoidal platelet deposition was observed in the hepatic tumors of humans and mice. Podoplanin was expressed in some of the Kupffer cells in the liver of the transgenic mice, indicating that platelet activation occurred via the interaction of podoplanin with C-type lectin receptor 2 (CLEC-2) on the platelet membrane. Additionally, erythrocyte dyscrasia and glomerulonephropathy/interstitial pneumonia associated with platelet deposition were observed. In the transgenic mice, aspirin (Asp) administration prevented platelet activation, reduced the liver/body weight ratio, decreased the platelet deposition in the liver, kidney, and lung, and prevented erythrocyte dyscrasia and ameliorated the renal/pulmonary changes. Thrombopoietin overproduction by BRAFV600E-mutated hepatocytes may contribute to hepatocyte proliferation via thrombocytosis, platelet activation, and the interaction of platelets with hepatic sinusoidal cells, while hematologic, renal, and pulmonary disorders due to aberrant platelet activation may lead to spontaneous death in the transgenic mice.


Assuntos
Carcinogênese/genética , Neoplasias Hepáticas Experimentais/patologia , Fígado/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Trombopoetina/metabolismo , Animais , Biópsia , Plaquetas/patologia , Medula Óssea/patologia , Capilares/patologia , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Proliferação de Células/genética , Dietilnitrosamina/administração & dosagem , Dietilnitrosamina/toxicidade , Feminino , Regulação Neoplásica da Expressão Gênica , Hepatectomia , Hepatócitos/patologia , Humanos , Fígado/irrigação sanguínea , Fígado/citologia , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Ativação Plaquetária/genética , Cultura Primária de Células , Proteínas Proto-Oncogênicas B-raf/metabolismo , Células Tumorais Cultivadas
11.
Anticancer Res ; 39(7): 3463-3467, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262870

RESUMO

BACKGROUND/AIM: Patient-derived orthotopic xenograft (PDOX) models have patient-like clinical features and may be imaged, in case of some cancers, by passaging of the tumors through transgenic nude mice expressing red-fluorescent protein (RFP) where they stably acquire RFP expressing stroma. The aim of the present study was to quantify red fluorescent area and intensity in colon-cancer peritoneal metastases in PDOX models in non-transgenic nude mice after passage in RFP transgenic nude mice by non-invasive external fluorescence imaging. MATERIALS AND METHODS: Tumor fragments originating from a colon cancer patient with peritoneal metastases were implanted in transgenic RFP nude mice. Resultant tumors were harvested, and fragments were implanted in the same strain a second time. Passaged tumors stably acquired RFP-expressing stroma from their transgenic hosts. The tumor with RFP-expressing stromal cells were harvested and implanted orthotopically in non-transgenic nude mice. At eight weeks post-implantation, non-invasive external RFP images were obtained. RFP area and intensity were measured and correlated with tumor weight and volume. RESULTS: Metastatic patient colon cancer can be stably and brightly labeled by passage in transgenic RFP-expressing nude mice such that tumor growth could be non-invasively imaged. Tumor growing could be non-invasively imaged when passaged to non-transgenic nude mice. A strong correlation between fluorescence intensity and area values with tumor weight and volume were established by external fluorescence imaging. CONCLUSION: This new tumor model of metastatic colon cancer can be used to evaluate novel therapeutics in real time for this recalcitrant disease.


Assuntos
Neoplasias do Colo/metabolismo , Proteínas Luminescentes/metabolismo , Neoplasias Peritoneais/metabolismo , Células Estromais/metabolismo , Animais , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/patologia , Neoplasias do Colo/secundário , Humanos , Camundongos Nus , Camundongos Transgênicos , Imagem Óptica , Neoplasias Peritoneais/diagnóstico por imagem , Neoplasias Peritoneais/etiologia , Neoplasias Peritoneais/patologia , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Anticancer Res ; 39(7): 3651-3660, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262891

RESUMO

BACKGROUND/AIM: Cytochrome P450 epoxygenase is a major enzyme involved in the metabolism of ω-3 polyunsaturated fatty acids (PUFAs) to produce biologically active ω-3 epoxy fatty acids (ω-3 epoxides). In general, all epoxy PUFAs including ω-3 epoxides are quickly metabolized/inactivated by soluble epoxide hydrolase (sEH) to form diol products. The aims of this study were to determine the effect and mechanism of fat-1 transgene, and ω-3 PUFA combined with sEH gene knockout or inhibitor on inhibiting pancreatic cancer and the related mechanisms involved. MATERIALS AND METHODS: PK03-mutant KrasG12D murine pancreatic carcinoma cells were inoculated into mouse models including fat-1, sEH-/- and C57BL/6J mice. The mice were fed with AIN-76A diet with or without ω-3 PUFA supplementation or treated with sEH inhibitor. In addition to tumor growth (tumor size and weight), cell proliferation, mutant Kras-mediated signaling, inflammatory reaction and angiogenesis were analyzed immunohisto-chemically and by western blot assay. ω-3 PUFA metabolism, particularly focusing on ω-3 epoxy fatty acids (ω-3 epoxides), was measured using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach. RESULTS: Significant decreases of weight and size of the PK03 pancreatic carcinoma were observed in the fat-1 transgenic mice treated with sEH inhibitor compared to those of C57BL/6J control mice fed with AIN-76A diet (weight: 0.28±0.04 g vs. 0.58±0.06 g; size: 187.0±17.5 mm3 vs. 519.3±60.6 mm3). In a separate experiment, sEH-/- mice fed ω-3 PUFA supplement and C57BL/6J mice treated with sEH inhibitor and fed ω-3 PUFA supplement exhibited a significant reduction in the weight and size of the pancreatic carcinoma compared to C57BL/6J control mice (weight: 0.26±.26 g and 0.39±.39 g vs. 0.69±0.11 g, respectively; size: 274.2±36.2 mm3 and 296.4±99.8 mm3 vs. 612.6±117.8 mm3, respectively). Moreover, compared to the pancreatic tumors in C57BL/6J control mice, the tumors in fat-1 transgenic mice treated with sEH inhibitor showed a significant less inflammatory cell infiltrate (62.6±9.2/HPF (high power field) vs. 8.0±1.2/HPF), tumor cell proliferation (48.5±1.7% vs. 16.5±1.6%), and angiogenesis (micro-vessel density (MVD): 35.0±1.0 vs. 11.1±0.5) immunohistochemically, as well as significantly increased caspase-3 labeled apoptosis (0.44±0.06% vs. 0.69±0.06%, respectively). Using western blot approach, significant inhibition of mutant Kras-activated signals including phosphorylated Serine/threonine kinases (cRAF), Mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK) were identified in pancreatic carcinoma of fat-1 transgenic mice treated with sEH inhibitor. Eicosanoic acid metabolic profiling of the serum specimens detected a significant increase of the ratios of epoxides to dihydroxy fatty acid (DiHDPE) for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and epoxides/dihydroxy octadecenoic acid (DiHOME) for arachidonic acid (ARA) and linoleic acid (LA), as well as a significant increase of epoxy metabolites of DHA, EPA, ARA and LA in fat-1 transgenic mice treated with a sEH inhibitor. CONCLUSION: ω-3 epoxy products from ω-3 PUFA metabolism play a crucial role in inhibiting pancreatic cancer growth, and use of ω-3 PUFAs combined with sEH inhibition is a strategy with high potential for pancreatic cancer treatment and prevention.


Assuntos
Adenocarcinoma/terapia , Proteínas de Caenorhabditis elegans/genética , Suplementos Nutricionais , Compostos de Epóxi/farmacologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-3/farmacologia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/patologia
13.
Nat Commun ; 10(1): 2901, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263101

RESUMO

Dysregulation of histone modifications promotes carcinogenesis by altering transcription. Breast cancers frequently overexpress the histone methyltransferase EZH2, the catalytic subunit of Polycomb Repressor Complex 2 (PRC2). However, the role of EZH2 in this setting is unclear due to the context-dependent functions of PRC2 and the heterogeneity of breast cancer. Moreover, the mechanisms underlying PRC2 overexpression in cancer are obscure. Here, using multiple models of breast cancer driven by the oncogene ErbB2, we show that the tyrosine kinase c-Src links energy sufficiency with PRC2 overexpression via control of mRNA translation. By stimulating mitochondrial ATP production, c-Src suppresses energy stress, permitting sustained activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which increases the translation of mRNAs encoding the PRC2 subunits Ezh2 and Suz12. We show that Ezh2 overexpression and activity are pivotal in ErbB2-mediated mammary tumourigenesis. These results reveal the hitherto unknown c-Src/mTORC1/PRC2 axis, which is essential for ErbB2-driven carcinogenesis.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Epigênese Genética , Complexo Repressor Polycomb 2/genética , Receptor ErbB-2/metabolismo , Quinases da Família src/metabolismo , Trifosfato de Adenosina/metabolismo , Adulto , Animais , Neoplasias da Mama/patologia , Carcinogênese , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Biossíntese de Proteínas , Receptor ErbB-2/genética , Quinases da Família src/genética
14.
Nat Commun ; 10(1): 2735, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227713

RESUMO

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer.


Assuntos
Interleucina-33/imunologia , Macrófagos/imunologia , Mastócitos/imunologia , Neoplasias Gástricas/imunologia , Aminopiridinas/administração & dosagem , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Cromolina Sódica/administração & dosagem , Modelos Animais de Doenças , Epitélio/imunologia , Epitélio/patologia , Feminino , Mucosa Gástrica/citologia , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pirróis/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise Serial de Tecidos , Microambiente Tumoral/imunologia
15.
Sheng Wu Gong Cheng Xue Bao ; 35(6): 1041-1049, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31232000

RESUMO

Nerve growth factor (NGF) can promote the development, differentiation and regeneration of neurons. Recently, in order to efficiently produce human NGF (hNGF) drugs with better efficacy, we created transgenic mice expressing hNGF specifically in their salivary glands, and purified highly active hNGF protein from their saliva. Some studies reported that the NGF secretion in mouse saliva is affected by gender and age. Here, in order to select hNGF transgenic mice with high NGF secretion for saliva collection and hNGF purification, we divided transgenic mice into 4 groups, including 28-day-old young males and females, 63-day-old adult males and females. We compared their saliva volume, total salivary protein amount, salivary mNGF protein amount and salivary hNGF protein amount. The results showed that the saliva volume as well as amounts of total salivary protein, salivary mNGF protein and salivary hNGF protein secreted by 63-day-old transgenic mice were significantly higher than those secreted by sex-match 28-day-old transgenic mice, and the salivary hNGF protein amount secreted by male transgenic mice at the age of 63 days was significantly higher than that of female transgenic mice at the same age; Among 4 groups of mice, 63-day-old male transgenic mice secreted the highest salivary hNGF content, which was about 46 times higher than that secreted by the 28-day-old female transgenic mice. Therefore, 63-day-old male transgenic mice should be selected for saliva collection and hNGF purification.


Assuntos
Saliva , Animais , Diferenciação Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fator de Crescimento Neural
16.
Clin Interv Aging ; 14: 817-826, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190768

RESUMO

Background: Alzheimer's disease (AD) is the most common neurodegenerative disorder that also involves neuroinflammation in addition to many other features. Icariin (ICA) as one of the active ingredients of Chinese herbal medicine has the immunomodulating function. This study aimed to investigate the immunotherapeutic potential of ICA on AD. Methods: APP/PS1 mice and wild type C57BL/6 mice were subjected to orally ICA administration (60 mg/kg/d) for 8 months. Then, the ethological and biochemical experiments, such as Morris water maze assay, Aß ELISA, blood T cell flow cytometry, and plasma and brain cytokines array, were conducted to evaluate the effects of ICA administration. Results: ICA significantly improved spatial learning and memory retention in APP/PS1 mice. Long-term application of ICA could also reduce hippocampus Aß deposition, modulate the differentiation of CD4+ T cells, and modulate the release of inflammatory cytokines in plasma and brain tissue. Conclusion: ICA shows the neuroprotective effects via modulating the CD4+ T lymphocyte-related immuno-inflammatory responses in APP/PS1 mice and may be a promising drug against AD progression.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Flavonoides/farmacologia , Memória/efeitos dos fármacos , Aprendizagem Espacial/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cognição/fisiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Presenilina-1
17.
Artigo em Chinês | MEDLINE | ID: mdl-31245943

RESUMO

OBJECTIVE: To study the effect of ß-sheet blocking peptide H102 on the expression of AMPK-mTOR autophagy pathway-related protein in APP/PS1 double transgenic AD mice. METHODS: Thirty male APP/PS1 transgenic male AD mice of 6 months old were randomly divided into AD group and H102 intervention group, and C57BL/6J male mice of the same age were used as control group (n=15). The mice in the HF group were administered with 5 µl (5.8 mg/kg) of H102 polypeptide solution through the nasal cavity at the same time period, and the mice in the control group and the AD group were given the same amount of blank adjuvant solution daily. The memory recognition ability was tested by a new object recognition experiment 30 days after continuous administration. Immunohistochemistry and Western blot were used to detect the expressions of phosphorylated AMP-activated protein kinase(P-AMPK),phosphorylated mammalian target of rapamycin (P-mTOR) and ratio of LC32to LC31(LC3II/I )in brain tissue. RESULTS: Compared with the control group, the new object recognition index (RI) of the AD group was significantly lower (P<0.05), and the P-AMPK and LC3II/I ratios in the brain of the mice were significantly lower (P<0.05). The expression of P-mTOR protein was increased significantly (P<0.05). Compared with the AD group, the RI of the H102 intervention group was increased significantly (P<0.05), and the P-AMPK and LC3II/I ratios in the brain tissue of the mice were increased significantly (P<0.05). The expression of P-mTOR protein was decreased significantly (P<0.05). CONCLUSION: H102 can improve the recognition and memory ability of AD mice by activating the AMPK-mTOR autophagy-related pathway.


Assuntos
Doença de Alzheimer , Autofagia , Memória , Peptídeos , Serina-Treonina Quinases TOR , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Precursor de Proteína beta-Amiloide , Animais , Autofagia/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/farmacologia , Distribuição Aleatória , Serina-Treonina Quinases TOR/efeitos dos fármacos
18.
Genome Biol ; 20(1): 110, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31159854

RESUMO

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) plays a pivotal role in our understanding of cellular heterogeneity. Current analytical workflows are driven by categorizing principles that consider cells as individual entities and classify them into complex taxonomies. RESULTS: We devise a conceptually different computational framework based on a holistic view, where single-cell datasets are used to infer global, large-scale regulatory networks. We develop correlation metrics that are specifically tailored to single-cell data, and then generate, validate, and interpret single-cell-derived regulatory networks from organs and perturbed systems, such as diabetes and Alzheimer's disease. Using tools from graph theory, we compute an unbiased quantification of a gene's biological relevance and accurately pinpoint key players in organ function and drivers of diseases. CONCLUSIONS: Our approach detects multiple latent regulatory changes that are invisible to single-cell workflows based on clustering or differential expression analysis, significantly broadening the biological insights that can be obtained with this leading technology.


Assuntos
Redes Reguladoras de Genes , Genômica/métodos , Transcriptoma , Doença de Alzheimer/genética , Animais , Diabetes Mellitus Tipo 2/genética , Estudos de Viabilidade , Genes Essenciais , Humanos , Camundongos Transgênicos , Análise de Célula Única
19.
Dokl Biochem Biophys ; 485(1): 153-156, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201639

RESUMO

A multiplex PCR test system for identification of the regulatory sequences of genetic constructs for transformation (promotor, insulator, and terminator) in the Mus musculus genome and for transgenic animal selection by genotyping with horizontal agarose gel electrophoresis detection was developed. The proposed system was validated by genotyping mouse strains producing human lactoferrin, heat shock protein HSP 70, firefly luciferase, and lysozyme, which were obtained by microinjections of linearized DNA into murine zygote pronucleus with random transgene integration into the genome using the pBC1 plasmid for expression of the gene of interest in milk of transformed animals (milk expression vector kit).


Assuntos
Técnicas de Genotipagem , Lactoferrina , Leite/metabolismo , Reação em Cadeia da Polimerase Multiplex , Animais , Lactoferrina/genética , Lactoferrina/metabolismo , Camundongos , Camundongos Transgênicos
20.
Nat Neurosci ; 22(7): 1182-1195, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209381

RESUMO

Understanding the diversity of cell types in the brain has been an enduring challenge and requires detailed characterization of individual neurons in multiple dimensions. To systematically profile morpho-electric properties of mammalian neurons, we established a single-cell characterization pipeline using standardized patch-clamp recordings in brain slices and biocytin-based neuronal reconstructions. We built a publicly accessible online database, the Allen Cell Types Database, to display these datasets. Intrinsic physiological properties were measured from 1,938 neurons from the adult laboratory mouse visual cortex, morphological properties were measured from 461 reconstructed neurons, and 452 neurons had both measurements available. Quantitative features were used to classify neurons into distinct types using unsupervised methods. We established a taxonomy of morphologically and electrophysiologically defined cell types for this region of the cortex, with 17 electrophysiological types, 38 morphological types and 46 morpho-electric types. There was good correspondence with previously defined transcriptomic cell types and subclasses using the same transgenic mouse lines.


Assuntos
Conjuntos de Dados como Assunto , Neurônios/classificação , Córtex Visual/citologia , Potenciais de Ação , Animais , Forma Celular , Bases de Dados Factuais , Genes Reporter , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Transcriptoma , Córtex Visual/fisiologia
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