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1.
PLoS One ; 17(9): e0274607, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36108271

RESUMO

Mesenchymal stem cells can be obtained and multiplied from various sources and have a very high capacity to release exosomes. Exosomes are nano-sized extracellular vesicles containing biological signaling molecules. This study aimed to determine the effect of MSC-derived exosomes as a drug delivery system for paclitaxel in cervical cancer cells. In this study, human MSC were isolated from wharton jelly of umbilical cord tissue (WJ-MSC), and cells were characterized by CD44, CD90, CD105, and CD34 staining. Exosomes were released in WJ-MSC cells with serum-starved conditions for 48 hours, and particle sizes and structures were examined with zeta-sizer and TEM. In addition, exosomes CD9, CD63, and CD81 markers were checked by western blot. Paclitaxel was loaded into exosomes (Exo-PAC) by electroporation and then incubated with Hela cervical cancer cells for 24 hours. TGF-ß, SMAD, Snail, Slug, ß-catenin, Notch, Caspase-3, Caspase-9, Bax, Bcl-2 protein and gene expression levels were analyzed in Hela cells. As a result, low concentration Exo-PAC induced apoptosis, and suppressed epithelial-mesenchymal transition proteins in Hela cells. In this study, it has been demonstrated that WJ-MSCs can be used as drug delivery systems for cervical cancer if exosomes are produced scalably in the future.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Neoplasias do Colo do Útero , Geleia de Wharton , Apoptose , Caspase 3/metabolismo , Caspase 9/metabolismo , Portadores de Fármacos/metabolismo , Exossomos/metabolismo , Feminino , Células HeLa , Humanos , Células-Tronco Mesenquimais/metabolismo , Paclitaxel/metabolismo , Paclitaxel/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismo , Geleia de Wharton/metabolismo , Proteína X Associada a bcl-2/metabolismo , beta Catenina/metabolismo
2.
BMC Ophthalmol ; 22(1): 368, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114477

RESUMO

BACKGROUND: Elevated intraocular pressure (IOP) is the main risk factor for glaucoma, which might cause the activation of astrocytes in optic nerve head. To determine the effect of mechanical stretch on the astrocytes, we investigated the changes in cell phenotype, proteins of interest and signaling pathways under biaxial stretch. METHOD: The cultured astrocytes in rat optic nerve head were stretched biaxially by 10 and 17% for 24 h, respectively. Then, we detected the morphology, proliferation and apoptosis of the stretched cells, and performed proteomics analysis. Protein expression was analyzed by Isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. Proteins of interest and signaling pathways were screened using Gene Ontology enrichment analysis and pathway enrichment analysis, and the results were verified by western blot and the gene-chip data from Gene Expression Omnibus (GEO) database. RESULT: The results showed that rearrangement of the actin cytoskeleton in response to stimulation by mechanical stress and proliferation rate of astrocytes decreased under 10 and 17% stretch condition, while there was no significant difference on the apoptosis rate of astrocytes in both groups. In the iTRAQ quantitative experiment, there were 141 differential proteins in the 10% stretch group and 140 differential proteins in the 17% stretch group. These proteins include low-density lipoprotein receptor-related protein (LRP6), caspase recruitment domain family, member 10 (CARD10), thrombospondin 1 (THBS1) and tetraspanin (CD81). The western blot results of LRP6, THBS1 and CD81 were consistent with that of iTRAQ experiment. ANTXR2 and CARD10 were both differentially expressed in the mass spectrometry results and GEO database. We also screened out the signaling pathways associated with astrocyte activation, including Wnt/ß-catenin pathway, NF-κB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, ECM-receptor interaction, and transforming growth factor-ß (TGF-ß) signaling pathway. CONCLUSION: Mechanical stimulation can induce changes in cell phenotype, some proteins and signaling pathways, which might be associated with astrocyte activation. These proteins and signaling pathways may help us have a better understanding on the activation of astrocytes and the role astrocyte activation played in glaucomatous optic neuropathy.


Assuntos
Glaucoma , Disco Óptico , Animais , Astrócitos , Glaucoma/genética , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , NF-kappa B/metabolismo , NF-kappa B/farmacologia , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ratos , Trombospondina 1/metabolismo , Trombospondina 1/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Fatores de Crescimento Transformadores/metabolismo , Fatores de Crescimento Transformadores/farmacologia , beta Catenina/metabolismo , beta Catenina/farmacologia
3.
Cell Commun Signal ; 20(1): 144, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114543

RESUMO

BACKGROUND: Notch signaling is highly conserved and critically involved in cell differentiation, immunity, and survival. Activation of the Notch pathway modulates immune cell functions during the inflammatory response. However, it remains unknown whether and how the macrophage Notch1 may control the innate immune signaling TAK1, and RIPK3-mediated hepatocyte necroptosis in liver ischemia and reperfusion injury (IRI). This study investigated the molecular mechanisms of macrophage Notch1 in modulating TAK1-mediated innate immune responses and RIPK3 functions in liver IRI. METHODS: Myeloid-specific Notch1 knockout (Notch1M-KO) and floxed Notch1 (Notch1FL/FL) mice (n = 6/group) were subjected to 90 min partial liver warm ischemia followed by 6 h of reperfusion. In a parallel in vitro study, bone marrow-derived macrophages (BMMs) were isolated from these conditional knockout mice and transfected with CRISPR/Cas9-mediated ß-catenin knockout (KO) vector followed by LPS (100 ng/ml) stimulation. RESULTS: IR stress-induced Notch1 activation evidenced by increased nuclear Notch intracellular domain (NICD) expression in liver macrophages. Myeloid Notch1 deficiency exacerbated IR-induced liver damage, with increased serum ALT levels, macrophage/neutrophil accumulation, and proinflammatory cytokines/chemokines production compared to the Notch1FL/FL controls. Unlike in the Notch1FL/FL controls, Notch1M-KO enhanced TRAF6, TAK1, NF-κB, RIPK3, and MLKL but reduced ß-catenin activation in ischemic livers. However, adoptive transfer of lentivirus ß-catenin-modified macrophages markedly improved liver function with reduced TRAF6, p-TAK1, RIPK3 and p-MLKL in IR-challenged livers. Moreover, disruption of RIPK3 in Notch1M-KO mice with an in vivo mannose-mediated RIPK3 siRNA delivery system diminished IR-triggered hepatocyte death. In vitro studies showed that macrophage NICD and ß-catenin co-localized in the nucleus, whereby ß-catenin interacted with NICD in response to LPS stimulation. Disruption of ß-catenin with a CRISPR/Cas9-mediated ß-catenin KO in Notch1FL/FL macrophage augmented TRAF6 activation leading to enhanced TAK1 function. While CRISPR/Cas9-mediated TRAF6 KO in Notch1M-KO macrophage inhibited RIPK3-mediated hepatocyte necroptosis after co-culture with primary hepatocytes. CONCLUSIONS: Macrophage Notch1 controls TAK1-mediated innate immune responses and RIPK3-mediated hepatocyte necroptosis through activation of ß-catenin. ß-catenin is required for the macrophage Notch1-mediated immune regulation in liver IRI. Our findings demonstrate that the macrophage Notch1-ß-catenin axis is a crucial regulatory mechanism in IR-triggered liver inflammation and provide novel therapeutic potential in organ IRI and transplant recipients. Video abstract.


Assuntos
Necroptose , Traumatismo por Reperfusão , Animais , Citocinas/metabolismo , Hepatócitos/metabolismo , Isquemia/metabolismo , Lipopolissacarídeos , Fígado/metabolismo , Macrófagos/metabolismo , Manose/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores , Traumatismo por Reperfusão/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , beta Catenina/metabolismo
4.
Acta Neuropathol Commun ; 10(1): 138, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114575

RESUMO

Non-functioning pituitary tumours (NF-PitNETs) are common intracranial benign neoplasms that can exhibit aggressive behaviour by invading neighbouring structures and, in some cases, have multiple recurrences. Despite resulting in severe co-morbidities, no predictive biomarkers of recurrence have been identified for NF-PitNETs. In this study we have used high-throughput mass spectrometry-based analysis to examine the phosphorylation pattern of different subsets of NF-PitNETs. Based on histopathological, radiological, surgical and clinical features, we have grouped NF-PitNETs into non-invasive, invasive, and recurrent disease groups. Tumour recurrence was determined based on regular clinical and radiological data of patients for a mean follow-up of 10 years (SD ± 5.4 years). Phosphoproteomic analyses identified a unique phosphopeptide enrichment pattern which correlates with disease recurrence. Candidate phosphorylated proteins were validated in a large cohort of NF-PitNET patients by western blot and immunohistochemistry. We identified a cluster of 22 phosphopeptides upregulated in recurrent NF-PitNETs compared to non-invasive and invasive subgroups. We reveal significant phosphorylation of the ß-catenin at Ser552 in recurrent and invasive NF-PitNETs, compared to non-invasive/non-recurrent NF-PitNET subgroup. Moreover, ß-catenin pSer552 correlates with the recurrence free survival among 200 patients with NF-PitNET. Together, our results suggest that the phosphorylation status of ß-catenin at Ser552 could act as potential biomarker of tumour recurrence in NF-PitNETs.


Assuntos
Tumores Neuroendócrinos , Neoplasias Hipofisárias , Humanos , Recidiva Local de Neoplasia , Tumores Neuroendócrinos/metabolismo , Fosfopeptídeos/metabolismo , Fosforilação , Neoplasias Hipofisárias/metabolismo , beta Catenina/metabolismo
5.
Stem Cell Res Ther ; 13(1): 443, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-36056439

RESUMO

BACKGROUND: Bone is a rigid organ that provides physical protection and support to vital organs of the body. Bone loss disorders are commonly associated with increased bone marrow adipose tissue. Bone marrow mesenchymal stromal/stem cells (BMSCs) are multipotent progenitors that can differentiate into osteoblasts, adipocytes, and chondrocytes. Cell division cycle 20 (CDC20) is a co-activator of anaphase promoting complex/cyclosome (APC/C), and is required for ubiquitin ligase activity. Our previous study showed that CDC20 promoted the osteogenic commitment of BMSCs and Cdc20 conditional knockout mice suggested a decline in bone mass. In this study, we found that knockdown of CDC20 promoted adipogenic differentiation of BMSCs by modulating ß-catenin, which suggested a link between adipogenesis and osteogenesis. METHODS: Lentivirus containing a CDC20 shRNA was used for CDC20 knockdown in human BMSCs (hBMSCs). Primary mouse BMSCs (mBMSCs) were isolated from Cdc20f/f and Sp7-Cre;Cdc20f/f mice. Adipogenesis was examined using quantitative real-time reverse transcription PCR (qRT-PCR) and western blotting analysis of adipogenic regulators, Oil Red O staining, and transplantation into nude mice. CDC20 knockout efficiency was determined through immunochemistry, qRT-PCR, and western blotting of bone marrow. Accumulation of adiposity was measured through histology and staining of bone sections. Exploration of the molecular mechanism was determined through western blotting, Oil Red O staining, and qRT-PCR. RESULTS: CDC20 expression in hBMSCs was significantly decreased during adipogenic differentiation. CDC20 knockdown enhanced hBMSC adipogenic differentiation in vitro. CDC20-knockdown hBMSCs showed more adipose tissue-like constructs upon hematoxylin and eosin (H&E) and Oil Red O staining. Sp7-Cre;Cdc20f/f mice presented increased adipocytes in their bone marrow compared with the control mice. mBMSCs from Sp7-Cre;Cdc20f/f mice showed upregulated adipogenic differentiation. Knockdown of CDC20 led to decreased ß-catenin levels, and a ß-catenin pathway activator (lithium chloride) abolished the role of CDC20 in BMSC adipogenic differentiation. CONCLUSIONS: Our findings showed that CDC20 knockdown enhanced adipogenesis of hBMSC and mBMSCs adipogenesis in vitro and in vivo. CDC20 regulates both adipogenesis and osteogenesis of BMSCs, and might lead to the development of new therapeutic targets for "fatty bone" and osteoporosis.


Assuntos
Adipogenia , Proteínas Cdc20/metabolismo , Células-Tronco Mesenquimais , Animais , Medula Óssea/metabolismo , Células da Medula Óssea , Proteínas Cdc20/genética , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Osteogênese/genética , beta Catenina/genética , beta Catenina/metabolismo
6.
Bioorg Chem ; 128: 106116, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36063753

RESUMO

Carnosic acid could disrupt the ß-catenin/BCL9 protein-protein interaction and inhibit ß-catenin dependent transcription, thereby reduce the incidence of colorectal cancer induced by abnormal activation of Wnt/ß-catenin signalling pathway. However, its activity was weak (IC50 for SW480: 28.2 ± 2.05 µM) and total synthesis was difficult. During the structural simplification of natural products, S0 was revealed to be the basic pharmacophore of carnosic acid. Subsequent structural optimization of S0 led to the discovery of S11 as a possible anticancer agent with prominent proliferation inhibition effect (IC50 for SW480: 9.56 ± 0.91 µM) and best selectivity index (SI = 3.0) against Wnt hyperactive cancer cells. Futher mechanism investigation through TOP/FOP dual luciferase reporter assay, immunofluorescence, co-immunoprecipitation, microscale thermophoresis, downstream oncoprotein expression and cell apoptosis showed that compound S11 could significantly inhibit the proliferation of SW480 cells via obvioudsly decreasing the nucleus translocation of ß-catenin and effectively disrupting ß-catenin/BCL9 protein-protein interaction. Additionally, cell migration, molecule docking, in vitro stability and solubility assays were also conducted. Overall, S11 was worthy of in-depth study as a potential inhibitor for the Wnt/ß-catenin pathway and its discovery also proved that the structural simplification of natural products was still one of the effective methods to find new lead compounds or candidate drugs.


Assuntos
Produtos Biológicos , beta Catenina , Androstenóis , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Hidroxibenzoatos , Via de Sinalização Wnt , beta Catenina/metabolismo
7.
Proc Natl Acad Sci U S A ; 119(37): e2203557119, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36067312

RESUMO

Developmental dysplasia of the hip (DDH) is one of the most common congenital skeletal malformations; however, its etiology remains unclear. Here, we conducted whole-exome sequencing in eight DDH families followed by targeted sequencing of 68 sporadic DDH patients. We identified likely pathogenic variants in the LRP1 (low-density lipoprotein receptor-related protein 1) gene in two families and seven unrelated patients. All patients harboring the LRP1 variants presented a typical DDH phenotype. The heterozygous Lrp1 knockout (KO) mouse (Lrp1+/-) showed phenotypes recapitulating the human DDH phenotypes, indicating Lrp1 loss of function causes DDH. Lrp1 knockin mice with a missense variant corresponding to a human variant identified in DDH (Lrp1R1783W) also presented DDH phenotypes, which were milder in heterozygotes and severer in homozygotes than those of the Lrp1 KO mouse. The timing of triradiate cartilage development was brought forward 1 or 2 wk earlier in the LRP-deficient mice, which leads to malformation of the acetabulum and femoral head. Furthermore, Lrp1 deficiency caused a significant decrease of chondrogenic ability in vitro. During the chondrogenic induction of mice bone marrow stem cells and ATDC5 (an inducible chondrogenic cell line), Lrp1 deficiency caused decreased autophagy levels with significant ß-catenin up-regulation and suppression of chondrocyte marker genes. The expression of chondrocyte markers was rescued by PNU-74654 (a ß-catenin antagonist) in an shRNA-Lrp1-expressed ATDC5 cell. Our study reveals a critical role of LRP1 in the etiology and pathogenesis of DDH, opening an avenue for its treatment.


Assuntos
Autofagia , Condrócitos , Displasia do Desenvolvimento do Quadril , Heterozigoto , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Animais , Autofagia/genética , Condrócitos/metabolismo , Condrócitos/patologia , Displasia do Desenvolvimento do Quadril/genética , Displasia do Desenvolvimento do Quadril/patologia , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Knockout , beta Catenina/metabolismo
8.
Oxid Med Cell Longev ; 2022: 9029544, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36105480

RESUMO

PHGDH attaches importance to serine biosynthesis in cancer cells and maintaining mitochondrial redox homeostasis. However, the role of PHGDH inhibitor CBR-5884 in cell ROS level and its downstream pathways has not been explored in epithelial ovarian cancer. Thus, we investigated the function and possible mechanism of PHGDH inhibitor CBR-5884 on epithelial ovarian cancer in vitro and in vivo. A2780, OVCAR3, and ES-2 were treated with CBR-5884 at different concentrations or different time points. Results showed that CBR-5884 inhibited epithelial ovarian cancer cell proliferation, migration, and invasion and increases cell ROS level. Meanwhile, CBR-5884 exerts antitumor effect through activating ROS/Wnt/ß-catenin pathway. Besides, CBR-5884 exerts antitumor effect in vivo. What's more, we explored the effect of CBR-5884 with or without PARP inhibitor Olaparib, which showed that the two together had a larger effect. In conclusion, PHGDH inhibitor CBR-5884 inhibits epithelial ovarian cancer proliferation, migration, and invasion through activating ROS/Wnt/ß-catenin pathway and plays a synergistic role with PARP inhibitor olaparib, which provided a theoretical basis for PHGDH inhibitor CBR-5884 in clinical treatment.


Assuntos
Antineoplásicos , Neoplasias Ovarianas , Antineoplásicos/farmacologia , Apoptose , Carcinoma Epitelial do Ovário/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/patologia , Ftalazinas , Piperazinas , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Espécies Reativas de Oxigênio/uso terapêutico , beta Catenina/metabolismo
9.
Development ; 149(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36098369

RESUMO

Neurovascular unit and barrier maturation rely on vascular basement membrane (vBM) composition. Laminins, a major vBM component, are crucial for these processes, yet the signaling pathway(s) that regulate their expression remain unknown. Here, we show that mural cells have active Wnt/ß-catenin signaling during central nervous system development in mice. Bulk RNA sequencing and validation using postnatal day 10 and 14 wild-type versus adenomatosis polyposis coli downregulated 1 (Apcdd1-/-) mouse retinas revealed that Lama2 mRNA and protein levels are increased in mutant vasculature with higher Wnt/ß-catenin signaling. Mural cells are the main source of Lama2, and Wnt/ß-catenin activation induces Lama2 expression in mural cells in vitro. Markers of mature astrocytes, including aquaporin 4 (a water channel in astrocyte endfeet) and integrin-α6 (a laminin receptor), are upregulated in Apcdd1-/- retinas with higher Lama2 vBM deposition. Thus, the Wnt/ß-catenin pathway regulates Lama2 expression in mural cells to promote neurovascular unit and barrier maturation.


Assuntos
Via de Sinalização Wnt , beta Catenina , Animais , Camundongos , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
10.
Diagn Pathol ; 17(1): 69, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36096860

RESUMO

OBJECTIVES: To investigate lymphoid enhancer factor 1 (LEF-1) protein expression in medulloblastomas (MBs) and its correlation with molecular grouping of MBs. METHODS: Expressions of LEF-1 and ß-catenin were detected by immunohistochemistry, and molecular grouping was performed based on the NanoString and sequencing techniques for 30 MBs. RESULTS: By genetic defining, 3 MBs were WNT-activated, 11 were SHH-activated, 3 were in Group 3 and 13 in Group 4 respectively. Nuclear LEF-1 staining was found in 8 MBs using immunohistochemical method. Three out of 8 showed diffuse and strong nuclear LEF-1 staining which were proved to be WNT-activated genetically, while the other 5 MBs with focal staining were SHH-activated genetically. The expression of LEF-1 protein was significantly correlated with genetically defined WNT-activated MBs (P < 0.0001). We also found focal nuclear ß-catenin expression ( less than 1% of tumor cells) in 5 MBs. LEF-1 positivity was significantly correlated nuclear ß-catenin expression (p < 0.001). CONCLUSIONS: Immunohistochemical staining of LEF-1 can be used as a supplement for ß-catenin to diagnosis WNT-activated Medulloblastomas, when ß-catenin is difficult to recognize for its cytoplasm/membrane staining background. Diffuse nuclear staining of LEF-1 indicates WNT-activated MB.


Assuntos
Neoplasias Cerebelares , Meduloblastoma , Biomarcadores , Neoplasias Cerebelares/diagnóstico , Humanos , Meduloblastoma/diagnóstico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Coloração e Rotulagem , Fatores de Transcrição TCF , beta Catenina/metabolismo
11.
Int J Mol Sci ; 23(17)2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36077403

RESUMO

Cashmere is initiated and develops in the fetal stages and the number and density of secondary hair follicles (SHFs) determine cashmere production and quality. Growing evidence indicates that both microRNA (miRNA) and long non-coding RNA (lncRNA) play an indispensable role in hair follicle (HF) growth and development. However, little is known about miRNAs, lncRNAs, and their functions as well as their interactions during cashmere initiation and development. Here, based on lncRNA and miRNA high-throughput sequencing and bioinformatics analysis, we identified 10,485 lncRNAs, 40,639 mRNAs, and 605 miRNAs in cashmere goat skin during HF induction, organogenesis, and cytodifferentiation stages. Among them, 521 lncRNAs, 5976 genes, and 204 miRNAs were differentially expressed (DE). KEGG analysis of DE genes indicated that ECM-receptor interaction and biosynthesis of amino acids were crucial for HF development. Notch, TGF-beta, and Wnt signaling pathways were also identified, which are conventional pathways associated with HF growth and development. Then, the ceRNA regulatory network was constructed, and the impact of lncRNA H19 was investigated in dermal papilla (DP) cells. The MTT, CCK-8, and EdU assays showed that the viability and proliferation of DP cells were promoted by H19, and mechanistic studies suggested that H19 performed its function through the chi-miR-214-3p/ß-catenin axis. The present study created a resource for lncRNA, miRNA, and mRNA studies in cashmere morphogenesis. It could contribute to a better understanding of the molecular mechanism of ncRNAs involved in the regulation of HF growth and development.


Assuntos
MicroRNAs , RNA Longo não Codificante , Animais , Proliferação de Células/genética , Perfilação da Expressão Gênica , Cabras/metabolismo , Folículo Piloso/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Organogênese , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , Transcriptoma , beta Catenina/genética , beta Catenina/metabolismo
12.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36077561

RESUMO

Wnt signaling plays an important role in embryogenesis and adult stem cell homeostasis. Its diminished activation is implicated in osteoporosis and degenerative neural diseases. However, systematic administration of Wnt-signaling agonists carries risk, as aberrantly activated Wnt/ß-catenin signaling is linked to cancer. Therefore, technologies for local modulation and control of Wnt signaling targeted to specific sites of disease or degeneration have potential therapeutic value in the treatment of degenerative diseases. We reported a facile approach to locally activate the canonical Wnt signaling cascade using nanomagnetic actuation or ligand immobilized platforms. Using a human embryonic kidney (HEK293) Luc-TCF/LEF reporter cell line, we demonstrated that targeting the cell membrane Wnt receptor, Frizzled 2, with peptide-tagged magnetic nanoparticles (MNPs) triggered canonical Wnt signaling transduction when exposed to a high-gradient, time-varying magnetic field, and the induced TCF/LEF signal transduction was shown to be avidity-dependent. We also demonstrated that the peptide retained signaling activity after functionalization onto glass surfaces, providing a versatile platform for drug discovery or recreation of the cell niche. In conclusion, these results showed that peptide-mediated Wnt signaling kinetics depended not only on ligand concentration but also on the presentation method of the ligand, which may be further modulated by magnetic actuation. This has important implications when designing future therapeutic platforms involving Wnt mimetics.


Assuntos
Nanopartículas de Magnetita , Via de Sinalização Wnt , Células HEK293 , Humanos , Ligantes , Peptídeos/farmacologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
13.
Biomed Res Int ; 2022: 3727165, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36046448

RESUMO

Noncoding RNAs were discovered to control a variety of developmental mechanisms, including osteogenesis. According to emerging evidence, cryptochrome circadian-regulating (CRY) proteins have emerged as essential controllers of osteoblast differentiation. The linked processes, on the other hand, are still unknown. The specific process that underpins osteoblast differentiation and proliferation is yet unknown. This research gives a meta-analysis of CRY2's impact on mouse osteoblast differentiation via the control of the WNT/ß-catenin signaling pathways. Western blot and quantitative real-time PCR were used to identify Cry2 expression levels, components in the osteoblast-associated signaling pathway, and osteoblast transcription markers. The osteogenic condition was measured utilizing alkaline phosphatase (ALP) and alizarin red (AR) staining, whereas cell growth rates were measured using CCK8 assays. An ectopic bone formation experiment was used to determine the osteogenic potential of osteoblasts. Cry2 stimulates the osteogenic development of mouse osteoblasts through canonical Wnt/ß-catenin signaling, according to the findings.


Assuntos
Via de Sinalização Wnt , beta Catenina , Animais , Diferenciação Celular/genética , Células Cultivadas , Criptocromos/genética , Camundongos , Osteoblastos/metabolismo , Osteogênese/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/genética , beta Catenina/metabolismo
14.
BMC Complement Med Ther ; 22(1): 233, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36056333

RESUMO

BACKGROUND: The plant-based medicinal food (PBMF) is a functional compound extracted from 6 medicinal and edible plants: Coix seed, L. edodes, A. officinalis L., H. cordata, Dandelion, and G. frondosa. Our previous studies have confirmed that the PBMF possesses anti-tumor properties in a subcutaneous xenograft model of nude mice. This study aims to further investigate the effects and potential molecular mechanisms of the PBMF on the recurrence and metastasis of gastric cancer (GC). METHODS: Postoperative recurrence and metastasis model of GC was successfully established in inbred 615 mice inoculated with mouse forestomach carcinoma (MFC) cells. After tumorectomy, 63 GC mice were randomly divided into five groups and respectively subject to different treatments for 15 days as below: model control group, 5-Fu group, and three doses of PBMF (43.22, 86.44, 172.88 g/kg PBMF in diet respectively). The inhibition rate (IR) of recurrence tumor weights and organ coefficients were calculated. Meanwhile, histopathological changes were examined and the metastasis IR in lungs and lymph node tissues was computed. The mRNA expressions related to the canonical Wnt/ß-catenin signaling pathway, epithelial-mesenchymal transition (EMT) and lymphangiogenesis were detected by RT-qPCR in recurrence tumors and/or lung tissues. Protein expressions of ß-catenin, p-ß-catenin (Ser33/37/Thr41), GSK-3ß, p-GSK-3ß (Ser9), E-cadherin, and Vimentin in recurrence tumors were determined by Western Blot. LYVE-1, VEGF-C/D, and VEGFR-3 levels in recurrence tumors and/or lung tissues were determined by immunohistochemistry staining. RESULTS: The mRNA, as well as protein expression of GSK-3ß were up-regulated and the mRNA expression of ß-catenin was down-regulated after PBMF treatment. Meanwhile, the ratio of p-ß-catenin (Ser33/37/Thr41) to ß-catenin protein was increased significantly and the p-GSK-3ß (Ser9) protein level was decreased. And PMBF could effectively decrease the mRNA and protein levels of Vimentin while increasing those of E-cadherin. Furthermore, PBMF markedly reduced lymphatic vessel density (LVD) (labeled by LYVE-1) in recurrence tumor tissues, and mRNA levels of VEGF-C/D, VEGFR-2/3 of recurrence tumors were all significantly lower in the high-dose group. CONCLUSIONS: PBMF had a significant inhibitory effect on recurrence and lung metastasis of GC. The potential mechanism may involve reversing EMT by inhabiting the Wnt/ß-catenin signaling pathway. Lymphatic metastasis was also inhibited by PBMF via down-regulating the activation of the VEGF-C/D-VEGFR-2/3 signaling cascade.


Assuntos
Neoplasias Pulmonares , Neoplasias Gástricas , Animais , Caderinas/farmacologia , Transição Epitelial-Mesenquimal , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/farmacologia , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , RNA Mensageiro , Neoplasias Gástricas/tratamento farmacológico , Fator C de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Vimentina/metabolismo , Vimentina/farmacologia , Via de Sinalização Wnt , beta Catenina/metabolismo
15.
Sci Rep ; 12(1): 15095, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064958

RESUMO

Phosphorylation controls important cellular signals and its dysregulation leads to disease. While most phospho-regulation studies are focused on kinases, phosphatases are comparatively overlooked. Combining peptide arrays with SAMDI mass spectrometry, we show that tyrosine phosphatase activity is restricted by basic amino acids adjacent to phosphotyrosines. We validate this model using two ß-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K). These mutants introduce a basic residue next to Y654, an established phosphorylation site where modification shifts ß-catenin from cell-cell adhesions and towards its essential nuclear role as Wnt-signaling effector. We show that T653-basic mutant ß-catenins are less efficiently dephosphorylated by phosphatases, leading to sustained Y654 phosphorylation and elevated Wnt signals, similar to those observed for Y654E phospho-mimic mutant mice. This model rationalizes how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6000+ potentially relevant cancer mutations.


Assuntos
Proteínas Tirosina Fosfatases , beta Catenina , Animais , Camundongos , Mutação , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Tirosina/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
16.
Cell Death Dis ; 13(9): 771, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36068223

RESUMO

Triggering receptors expressed on myeloid cells 2 (TREM2) is considered a protective factor to protect host from bacterial infection, while how it elicits this role is unclear. In the present study, we demonstrate that deficiency of triggering receptors expressed on myeloid cells 2 (TREM2) significantly enhanced macrophage pyroptosis induced by four common pyogenic bacteria including Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Escherichia coli. TREM2 deficiency also decreased bacterial killing ratio of macrophage, while Caspase-1 or GSDMD inhibition promoted macrophage-mediated clearance to these bacteria. Further study demonstrated that the effect of TREM2 on macrophage pyroptosis and bacterial eradication mainly dependents on the activated status of NLRP3 inflammasome. Moreover, as the key downstream of TREM2, ß-catenin phosphorylated at Ser675 by TREM2 signal and accumulated in nucleus and cytoplasm. ß-catenin mediated the effect of TREM2 on NLRP3 inflammasome and macrophage pyroptosis by reducing NLRP3 expression, and inhibiting inflammasome complex assembly by interacting with ASC. Collectively, TREM2/ß-catenin inhibits NLRP3 inflammasome to regulate macrophage pyroptosis, and enhances macrophage-mediated pyogenic bacterial clearance.


Assuntos
Inflamassomos , Piroptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pseudomonas aeruginosa , beta Catenina/metabolismo
17.
Genet Res (Camb) ; 2022: 5338956, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36072013

RESUMO

Objectives: Accumulating evidence indicates that the expression and/or variants of several genes play an essential role in the progress of colorectal cancer (CRC). The current study is a meta-analysis undertaken to estimate the prognosis and survival associated with CTNNB1/ß-catenin, APC, Wnt, SMAD3/4, TP53, and Cyclin D1 genes among CRC patients. Methods: The authors searched PubMed, EMBASE, and Science Direct for relevant reports published between 2000 and 2020 and analyzed them to determine any relationship between the (immunohistochemically/sequencing-detected) gene expression and variants of the selected genes and the survival of CRC patients. Results: The analysis included 34,074 patients from 64 studies. To evaluate association, hazard ratios (HRs) were estimated for overall survival (OS) or disease-free survival (DFS), with a 95% confidence interval (CIs). Pooled results showed that ß-catenin overexpression, APC mutation, SMAD-3 or 4 loss of expression, TP53 mutations, and Cyclin D1 expression were associated with shorter OS. ß-Catenin overexpression (HR: 0.137 (95% CI: 0.131-0.406)), loss of expression of SMAD3 or 4 (HR: 0.449 (95% CI: 0.146-0.753)), the mutations of TP53 (HR: 0.179 (95% CI: 0.126-0.485)), and Cyclin D1 expression (HR: 0.485 (95% CI: 0.772-0.198)) also presented risk for shorter DFS. Conclusions: The present meta-analysis indicates that overexpression or underexpression and variants of CTNNB1/ß-catenin, APC, SMAD3/4, TP53, and Cyclin D1 genes potentially acted as unfavorable biomarkers for the prognosis of CRC. The Wnt gene was not associated with prognosis.


Assuntos
Neoplasias Colorretais , beta Catenina , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Genes bcl-1 , Humanos , Prognóstico , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Supressora de Tumor p53/genética , beta Catenina/genética , beta Catenina/metabolismo
18.
Contrast Media Mol Imaging ; 2022: 5132691, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36082059

RESUMO

Objective: To explore the effect of the Wnt/ß-catenin signaling pathway on the proliferation and apoptosis of gastric cancer cells. Methods: An MTT colorimetric assay was used to detect the inhibitory effect of the Wnt/ß-catenin signaling pathway inhibitor FH535 on the proliferation of MKN45 gastric cancer cells. The cell proliferation index (PI) and apoptosis index (AI) were measured by flow cytometry. The expression levels of ß-catenin, c-myc, and cleaved caspase-3 in MKN45 gastric cancer cells were detected. Results: After using the Wnt/ß-catenin signaling pathway inhibitor FH535, MKN45 gastric cancer cells showed obvious shrinkage, death, and cell density decrease. MTT showed that the A value of MKN45 gastric cancer cells in FH535 group was significantly lower than that in the control group (P < 0.05). The survival rate of MKN45 gastric cancer cells in FH535 group was significantly lower than that in the control group (P < 0.05). The cell cycle of gastric cancer was arrested in G0/G1 phase. The expression levels of ß-catenin and c-myc protein in MKN45 gastric cancer cells in FH535 group decreased significantly (P < 0.05), while the expression level of cleaved caspase-3 protein increased significantly (P < 0.05). Conclusion: The Wnt/ß-catenin signal molecule can maintain the proliferation of gastric cancer cells. Inhibition of the Wnt/ß-catenin signaling pathway can inhibit the proliferation of gastric cancer cells and promote the apoptosis of MKN45 gastric cancer cells.


Assuntos
Neoplasias Gástricas , Via de Sinalização Wnt , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , beta Catenina/metabolismo , beta Catenina/farmacologia
19.
Allergol Immunopathol (Madr) ; 50(5): 75-83, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36086967

RESUMO

BACKGROUND: Asthma is a heterogeneous and complex chronic airway disease with a high incidence rate, characterized by chronic airway inflammation. Although the anti-inflammatory effect of zeaxanthin has been demonstrated in various disease models, its explicit role in allergic asthma remains elusive. METHODS: An allergic asthma model was established by ovalbumin (OVA) stimulation in BALB/c nude mice. The pathological examination, collagen deposition and expression of α-smooth muscle actin (α-SMA) in lung tissues were determined by hematoxylin and eosin (H&E), MASSON and immunofluorescence staining, respectively. Besides, the effect of zeaxanthin on inflammation and oxidative stress was assessed by the enzyme-linked immunosorbent assay (ELISA) and spectrophotometry measure. Moreover, the underlying mechanism was analyzed by detecting the expression of phosphorylated p38 (p-p38), p38, ß-catenin, p-c-Jun N-terminal kinase (p-JNK) and JNK with western blot assays. RESULTS: The distinct infiltration of inflammatory cells was observed in the OVA-induced asthma mice model with significantly increased concentrations of immunoglobulin E (IgE), interleukin-4 (IL-4), IL-5, IL-13 and eotaxin (p˂0.001), which were prominently reversed by zeaxanthin treatment (p˂0.001). In addition, zeaxanthin treatment decreased the OVA-induced collagen deposition and α-SMA expression. A similar inhibitory effect of zeaxanthin on the oxidative stress was also observed in the OVA-induced asthma mice model, as evidenced by the prominent decrease of malondialdehyde (MDA) concentration and the remarkable increase of superoxide dismutase (SOD), glutathione S transferase (GST) and Glutathione (GSH) concentrations (p˂0.001). Moreover, zeaxanthin introduction markedly reduced the relative expressions of p-p38/p38, ß-catenin and p-JNK/JNK in the OVA-induced asthma mice model (p˂0.001), indicating that zeaxanthin suppressed the p38 mitogen-activated protein kinase (p38 MAPK)/ß-catenin signaling pathway in the OVA-induced asthma mice model. CONCLUSIONS: Zeaxanthin attenuated OVA-induced allergic asthma in mice via modulating the p38 MAPK/ß-catenin signaling pathway.


Assuntos
Asma , beta Catenina , Animais , Asma/etiologia , Modelos Animais de Doenças , Inflamação/complicações , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ovalbumina/efeitos adversos , Transdução de Sinais , Zeaxantinas/efeitos adversos , beta Catenina/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
World J Gastroenterol ; 28(26): 3027-3046, 2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-36051330

RESUMO

Colorectal cancer (CRC) continues to be one of the main causes of death from cancer because patients progress unfavorably due to resistance to current therapies. Dysregulation of the Wnt/ß-catenin pathway plays a fundamental role in the genesis and progression of several types of cancer, including CRC. In many subtypes of CRC, hyperactivation of the ß-catenin pathway is associated with mutations of the adenomatous polyposis coli gene. However, it can also be associated with other causes. In recent years, studies of the tumor microenvironment (TME) have demonstrated its importance in the development and progression of CRC. In this tumor nest, several cell types, structures, and biomolecules interact with neoplastic cells to pave the way for the spread of the disease. Cross-communications between tumor cells and the TME are then established primarily through paracrine factors, which trigger the activation of numerous signaling pathways. Crucial advances in the field of oncology have been made in the last decade. This Minireview aims to actualize what is known about the central role of the Wnt/ß-catenin pathway in CRC chemoresistance and aggressiveness, focusing on cross-communication between CRC cells and the TME. Through this analysis, our main objective was to increase the understanding of this complex disease considering a more global context. Since many treatments for advanced CRC fail due to mechanisms involving chemoresistance, the data here exposed and analyzed are of great interest for the development of novel and effective therapies.


Assuntos
Neoplasias Colorretais , beta Catenina , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Microambiente Tumoral , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
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