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1.
Eur J Med Chem ; 216: 113311, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33677350

RESUMO

Drugs of targeting both activin receptor-like kinase 5 (ALK5) and p38α have therapeutic advantages, making them attractive treatment options for tumors. Two series of 4-(1H-indazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-imidazoles 13a-g and 4-(1-methyl-1H-indazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-imidazoles 20a-g were synthesized and evaluated for ALK5 and p38α mitogen-activated protein kinase inhibitory activity. The most potent compound, 13c (J-1090), inhibited ALK5- and p38α-mediated phosphorylation with half-maximal inhibitor concentrations of 0.004 µM and 0.004 µM, respectively, in the enzymatic assay. In this study, the effectiveness of 13c in transforming growth factor (TGF-ß)-exposed U87MG cells was investigated using western blotting, immunofluorescence assays, cell migration assay, invasion assay, and RT-PCR analysis. 13c inhibited the protein expression of Slug and the protein and RNA expression of the mesenchymal-related proteins N-cadherin and vimentin. Furthermore, 13c markedly suppressed TGF-ß-induced epithelial-to-mesenchymal transition (EMT), migration, and invasion in U87MG cells. These results suggest that 13c is a novel inhibitor of ALK5 with potential utility in the treatment of human glioma.


Assuntos
Imidazóis/química , Indazóis/química , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Sítios de Ligação , Caderinas/genética , Caderinas/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/farmacologia , Vimentina/genética , Vimentina/metabolismo
2.
Eur J Med Chem ; 214: 113188, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33550185

RESUMO

Herein, we describe the design, synthesis and structure-activity relationships of a series of novel s-triazine compounds can induce methuotic phenotype in various types of cancer cells. (E)-1-(4-Chlorophenyl)-3-(4-((4-morpholino-6-styryl-1,3,5-triazine-2-yl)amino)phenyl)urea, compound V6, exhibited a striking methuotic phenotype with a minimal effective concentration of less than 10 nM in U87 glioblastoma cells. Based on structure-activity relationship studies, we designed and synthesized an active probe P1 that retained the full potential of V6 in inducing the methuotic phenotype in U87 glioblastoma cells. Using this probe following affinity-based proteomic profiling strategy, we identified vimentin as the specific target protein of compound V6. Molecular docking revealed that V6 can form hydrogen bonds with vimentin at 273R and 276Y in its rod domain.


Assuntos
Antineoplásicos/farmacologia , Triazinas/farmacologia , Vimentina/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fenótipo , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/química , Células Tumorais Cultivadas , Vimentina/genética , Vimentina/metabolismo
3.
Anticancer Res ; 41(1): 169-174, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33419810

RESUMO

BACKGROUND/AIM: Vimentin3 (Vim3) was recently described as a tumour marker for the direct discrimination between benign and malignant kidney tumours. Here, we examined its expression in prostate cancer (PCa) cell lines and the regulation of its expression by endothelin receptors. MATERIALS AND METHODS: Prostate cancer cell lines (PC3, DU145, LNCap) were incubated with endothelin 1 (ET-1), BQ123 [endothelin A receptor (ETAR) antagonist], BQ788 [endothelin B receptor (ETBR) antagonist], BQ123+ET-1, BQ788+ET-1 for 24 h and a scratch assay was performed. Cell extracts were analysed by western blotting and qRT-PCR. RESULTS: ET-1 induced Vim3 overexpression. Blocking the ETBR in the different prostate cancer cell lines yielded a higher migration rate, whereby Vim3 expression was significantly increased. CONCLUSION: Vim3 concentration increases in cell lines without a functional ETBR and may be used as a marker for PCas where ETBR is frequently methylated.


Assuntos
Expressão Gênica , Neoplasias da Próstata/genética , Vimentina/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Endotelina-1/genética , Endotelina-1/metabolismo , Humanos , Masculino , Neoplasias da Próstata/metabolismo
4.
Gene ; 766: 145163, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32980450

RESUMO

BACKGROUND: Cardia adenocarcinoma (CA) is a distinct form of gastric cancer, and the optimal means of treating it remains controversial. At present, the role of the condensation complex gene non-SMC condensin I complex subunit G (NCAPG) in CA is uncertain, and as such the present study was designed to elucidate its importance in this oncogenic context. METHODS: We first used bioinformatics approaches to assess NCAPG expression profiles in CA using public databases. Protein profiling was also used to examine the expression of this protein in CA tumors and adjacent tissues from 20 patients. Then the expression of NCAPG in CA samples was quantified via qRT-PCR and Western blotting. NCAPG knockdown and overexpression in the SGC-7901 and AGS cell lines were subsequently performed, after which the expression of key proteins associated with epithelial-mesenchymal transition (EMT; E-cadherin, vimentin, N-cadherin, Snail, Slug) and the regulation of apoptotic responses (caspase-3, Bax, Bcl-2) was measured. The mechanistic role of NCAPG in CA was additionally studied by analyzing proteins associated with Wnt/ß-catenin signaling including Wnt1, phosphorylated GSK3ß, ß-catenin, and phosphorylated ß- catenin. The impact of NCAPG on the migration, survival, and invasion of CA cells was further examined. RESULTS: CA samples exhibited high NCAPG expression. When this gene was overexpressed in cell lines, it reduced caspase-3, Bax, and E-cadherin levels whereas it elevated Bcl-2, vimentin, N-cadherin, Snail, and Slug levels. NCAPG overexpression also resulted in the enhanced expression of Wnt1, phosphorylated GSK3ß, and total ß-catenin and the reduced expression of phosphorylated ß-catenin. The knockdown of NCAPG, in contrast, yielded the opposite phenotype. At a functional level, the overexpression of NCAPG improved the apoptotic resistance of CA cells while driving them to undergo EMT and to become more invasive and migratory. CONCLUSIONS: NCAPG overexpression can promote EMT and suppress tumor cell apoptosis via the activation of Wnt/ß-catenin signaling.


Assuntos
Adenocarcinoma/genética , Apoptose/genética , Cárdia/patologia , Proteínas de Ciclo Celular/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Gástricas/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Oncogenes/genética , Neoplasias Gástricas/patologia , Vimentina/genética
5.
Methods Mol Biol ; 2179: 353-383, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32939733

RESUMO

Metastasis results from the ability of cancer cells to grow and to spread beyond the primary tumor to distant organs. Epithelial-to-Mesenchymal Transition (EMT), a fundamental developmental process, is reactivated in cancer cells, and causes epithelial properties to evolve into mesenchymal and invasive ones. EMT changes cellular characteristics between two distinct states, yet, the process is not binary but rather reflects a broad spectrum of partial EMT states in which cells exhibit various degrees of intermediate epithelial and mesenchymal phenotypes. EMT is a complex multistep process that involves cellular reprogramming through numerous signaling pathways, alterations in gene expression, and changes in chromatin morphology. Therefore, expression of key proteins, including cadherins, occludin, or vimentin must be precisely regulated. A comprehensive understanding of how changes in nuclear organization, at the level of single genes clusters, correlates with these processes during formation of metastatic cells is still missing and yet may help personalized prognosis and treatment in the clinic. Here, we describe methods to correlate physiological and molecular states of cells undergoing an EMT process with chromatin rearrangements observed via FISH labeling of specific domains.


Assuntos
Transição Epitelial-Mesenquimal , Hibridização in Situ Fluorescente/métodos , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Hibridização in Situ Fluorescente/normas , Ocludina/genética , Ocludina/metabolismo , Sensibilidade e Especificidade , Vimentina/genética , Vimentina/metabolismo
6.
Biomed Pharmacother ; 133: 111068, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378968

RESUMO

Vimentin, a kind of intermediate filament protein III in mesenchymal cells, has become a highly researched topic around the world in recent years, as it holds complex biological functions and plays an important role in the epithelial-mesenchymal transition in the evolution of various tumors. This article reviews the biological function of vimentin and its relationship with breast cancer in order to provide novel ideas about the clinical diagnosis and targeted therapy of breast cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Vimentina/metabolismo , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Transdução de Sinais , Vimentina/genética
7.
Int J Mol Sci ; 21(24)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339388

RESUMO

Articular cartilage is a skeletal tissue of avascular nature and limited self-repair capacity. Cartilage-degenerative diseases, such as osteoarthritis (OA), are difficult to treat and often necessitate joint replacement surgery. Cartilage is a tough but flexible material and relatively easy to damage. It is, therefore, of high interest to develop methods allowing chondrocytes to recolonize, to rebuild the cartilage and to restore joint functionality. Here we studied the in vitro production of cartilage-like tissue using human articular chondrocytes exposed to the Random Positioning Machine (RPM), a device to simulate certain aspects of microgravity on Earth. To screen early adoption reactions of chondrocytes exposed to the RPM, we performed quantitative real-time PCR analyses after 24 h on chondrocytes cultured in DMEM/F-12. A significant up-regulation in the gene expression of IL6, RUNX2, RUNX3, SPP1, SOX6, SOX9, and MMP13 was detected, while the levels of IL8, ACAN, PRG4, ITGB1, TGFB1, COL1A1, COL2A1, COL10A1, SOD3, SOX5, MMP1, and MMP2 mRNAs remained unchanged. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis demonstrated among others the importance of these differentially regulated genes for cartilage formation. Chondrocytes grown in DMEM/F-12 medium produced three-dimensional (3D) spheroids after five days without the addition of scaffolds. On day 28, the produced tissue constructs reached up to 2 mm in diameter. Using specific chondrocyte growth medium, similar results were achieved within 14 days. Spheroids from both types of culture media showed the typical cartilage morphology with aggrecan positivity. Intermediate filaments form clusters under RPM conditions as detected by vimentin staining after 7 d and 14 d. Larger meshes appear in the network in 28-day samples. Furthermore, they were able to form a confluent chondrocyte monolayer after being transferred back into cell culture flasks in 1 g conditions showing their suitability for transplantation into joints. Our results demonstrate that the cultivation medium has a direct influence on the velocity of tissue formation and tissue composition. The spheroids show properties that make them interesting candidates for cellular cartilage regeneration approaches in trauma and OA therapy.


Assuntos
Cartilagem/citologia , Engenharia Tecidual/métodos , Simulação de Ausência de Peso/instrumentação , Cartilagem/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno/genética , Colágeno/metabolismo , Subunidades alfa de Fatores de Ligação ao Core/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Meios de Cultura/química , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Fatores de Transcrição SOX , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Engenharia Tecidual/instrumentação , Vimentina/genética , Vimentina/metabolismo
8.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(12): 1720-1725, 2020 Dec 30.
Artigo em Chinês | MEDLINE | ID: mdl-33380386

RESUMO

OBJECTIVE: To investigate the effects of ALKBH5 on migration, invasion and epithelial-mesenchymal transition (EMT) of human trophoblast cells. METHODS: The expression plasmid of ALKBH5 or a negative control plasmid (ALKBH5-NC) was transfected in human trophoblast HTR-8 /SVneo cells, and the expressions of ALKBH5 mRNA and protein were detected by qRT-PCR and Western blotting. Transwell assay was used to assess the changes in migration and invasion abilities of the trophoblast cells after the transfection. Western blotting was performed to detect the expressions of EMT-related proteins in the cells including vimentin, fibronectin, E-cadherin, N-cadherin, MMP9 and MMP2. RESULTS: ALKBH5 mRNA and protein expressions were significantly higher in ALKBH5 group than in the control group (P < 0.05). Over-expression of ALKBH5 significantly attenuated migration and invasion abilities of HTR-8/Svneo cells (P < 0.05). Compared with the control cells, the cells overexpressing ALKBH5 showed an up-regulated expression of E-cadherin and down-regulated expressions of vimentin, fibronectin, N-cadherin, MMP9 and MMP2 (P < 0.05). CONCLUSIONS: ALKBH5 is involved in the pathogenesis of preeclampsia by inhibiting EMT of trophoblast cells and hence reducing their migration and invasion abilities.


Assuntos
Pré-Eclâmpsia , Trofoblastos , Homólogo AlkB 5 da RNA Desmetilase , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Humanos , Gravidez , Vimentina/genética
9.
Nat Commun ; 11(1): 5127, 2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33046716

RESUMO

Despite the importance of AKT overactivation in tumor progression, results from clinical trials of various AKT inhibitors remain suboptimal, suggesting that AKT-driven tumor metastasis needs to be further understood. Herein, based on long non-coding RNA (lncRNA) profiling induced by active AKT, we identify that VAL (Vimentin associated lncRNA, LINC01546), which is directly induced by AKT/STAT3 signaling, functions as a potent pro-metastatic molecule and is essential for active AKT-induced tumor invasion, metastasis and anoikis resistance in lung adenocarcinoma (LAD). Impressively, chemosynthetic siRNAs against VAL shows great therapeutic potential in AKT overactivation-driven metastasis. Interestingly, similar to activated AKT in LAD cells, although unable to induce epithelial-mesenchymal transition (EMT), VAL exerts potent pro-invasive and pro-metastatic effects through directly binding to Vimentin and competitively abrogating Trim16-depedent Vimentin polyubiquitination and degradation. Taken together, our study provides an interesting demonstration of a lncRNA-mediated mechanism for active AKT-driven EMT-independent LAD metastasis and indicates the great potential of targeting VAL or Vimentin stability as a therapeutic approach.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Vimentina/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/fisiopatologia , Animais , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/fisiopatologia , Camundongos , Metástase Neoplásica , Proteólise , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Vimentina/genética
10.
Am J Surg Pathol ; 44(10): 1295-1307, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32675658

RESUMO

BACKGROUND: Secretory carcinoma (SC), originally described as mammary analogue SC, is a predominantly low-grade salivary gland neoplasm characterized by a recurrent t(12;15)(p13;q25) translocation, resulting in ETV6-NTRK3 gene fusion. Recently, alternative ETV6-RET, ETV6-MAML3, and ETV6-MET fusions have been found in a subset of SCs lacking the classic ETV6-NTRK3 fusion transcript, but still harboring ETV6 gene rearrangements. DESIGN: Forty-nine cases of SC revealing typical histomorphology and immunoprofile were analyzed by next-generation sequencing using the FusionPlex Solid Tumor kit (ArcherDX). All 49 cases of SC were also tested for ETV6, RET, and NTRK3 break by fluorescence in situ hybridization and for the common ETV6-NTRK3 fusions using reverse transcription polymerase chain reaction. RESULTS: Of the 49 cases studied, 37 (76%) occurred in the parotid gland, 7 (14%) in the submandibular gland, 2 (4%) in the minor salivary glands, and 1 (2%) each in the nasal mucosa, facial skin, and thyroid gland. SCs were diagnosed more frequently in males (27/49 cases; 55%). Patients' age at diagnosis varied from 15 to 80 years, with a mean age of 49.9 years. By molecular analysis, 40 cases (82%) presented the classic ETV6-NTRK3 fusion, whereas 9 cases (18%) revealed an alternate fusion. Of the 9 cases negative for the ETV6-NTRK3 fusion, 8 cases presented with ETV6-RET fusion. In the 1 remaining case in the parotid gland, next-generation sequencing analysis identified a novel VIM-RET fusion transcript. In addition, the analysis indicated that 1 recurrent high-grade case in the submandibular gland was positive for both ETV6-NTRK3 and MYB-SMR3B fusion transcripts. CONCLUSIONS: A novel finding in our study was the discovery of a VIM-RET fusion in 1 patient with SC of the parotid gland who could possibly benefit from RET-targeted therapy. In addition, 1 recurrent high-grade case was shown to harbor 2 different fusions, namely, ETV6-NTRK3 and MYB-SMR3B. The expanded molecular spectrum provides a novel insight into SC oncogenesis and carries important implications for molecular diagnostics, as this is the first SC-associated translocation with a non-ETV6 5' fusion partner. This finding further expands the definition of SC while carrying implications for selecting the appropriate targeted therapy.


Assuntos
Carcinoma Secretor Análogo ao Mamário/genética , Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias das Glândulas Salivares/genética , Vimentina/genética , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas e Peptídeos Salivares/genética , Adulto Jovem
11.
Environ Toxicol ; 35(10): 1050-1057, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32469461

RESUMO

Epithelial-to-mesenchymal transition (EMT) is key to invasion and metastasis by oral squamous carcinoma (OSCC) cells. MicroRNAs (miRNAs) such as miRNA-146a are known to be upregulated in OSCC. However, it is unclear whether they are involved in driving EMT. Here, we investigated the effect of miR-146a overexpression on proliferation, migration, and EMT in OSCC cells. OSCC cells were transfected with a plasmid expressing miR-146a precursor. Cell lines that stably overexpressed miRNA-146a were assessed for proliferation, colony formation, and invasiveness in vitro. Expression of markers and regulators of EMT, cell motility, and invasion were measured by qRT-PCR and western blot. Potential miRNA-146a binding sites in the 3'UTR of ST8SIA4 were identified by bioinformatic analysis. To confirm that miRNA-146a binds to and regulates ST8SIA4, we transfected OSCC cell lines with miRNA-146a mimics and a luciferase reporter construct containing either the wild type or mutant 3'UTR of ST8SIA4. OSCC cell lines that overexpressed miR-146a displayed higher proliferation, colony formation, invasion, and MMP-2 activity than cells transfected with a control vector. Overexpression of miR-146a also decreased expression of the epithelial cell marker E-cadherin and increased expression of Twist1, a transcription factor that promotes EMT, as well as markers associated with mesenchymal cells (vimentin and N-cadherin) and tumor invasion (p-paxillin and p-cortactin). Luciferase expression was lower in OSCC cells transfected with miRNA-146a mimics or with luciferase constructs carrying the wild type, but not mutant, 3'UTR of ST8SIA4. Overexpression of miR-146a promotes EMT phenotypes and may drive tumorigenesis and progression in OSCC, making it a useful target for future OSCC treatments.


Assuntos
Carcinoma de Células Escamosas/genética , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Neoplasias Bucais/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Bucais/patologia , Invasividade Neoplásica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plasmídeos , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Vimentina/genética
12.
J Clin Pathol ; 73(11): 748-753, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32366597

RESUMO

AIMS: L1 cell adhesion molecule (L1CAM) has been shown to be correlated with tumour progression, attributed to its possible association with epithelial-mesenchymal transition (EMT), characterised by the expression of vimentin and loss of e-cadherin. Herein, we investigate the associations between L1CAM and clinicopathological parameters, as well as the expression of vimentin and e-cadherin, in carcinomas restricted to the cervix. METHODS: The study was retrospective observational and included 45 squamous cell carcinomas (63.4%) and 26 adenocarcinomas (36.6%) submitted to primary surgical treatment. Patient age, FIGO (International Federation of Gynecology and Obstetrics) stage, tumour size and follow-up were obtained from the medical records. All the slides were revised to evaluate histological differentiation, lymphovascular space invasion, depth of infiltration, disease-free cervical wall thickness, pattern of invasion front, Silva pattern (for adenocarcinomas) and the percentage of tumour-infiltrating lymphocytes. Tissue microarrays were constructed for immunohistochemical staining for L1CAM, e-cadherin and vimentin. RESULTS: Adenocarcinomas were associated with lower disease-free and overall survival. L1CAM and vimentin expressions were more frequent among adenocarcinomas, although loss of e-cadherin expression was more common among squamous carcinomas. L1CAM expression was associated with larger tumours, vimentin expression and lower disease-free survival. No association was observed between the expression of either L1CAM or vimentin and loss of e-cadherin. High levels of tumour-infiltrating lymphocytes were more frequent in squamous cell carcinoma, high-grade tumours, destructive pattern at front of invasion and loss of e-cadherin expression. CONCLUSIONS: Our results confirm the prognostic role of L1CAM in cervical carcinomas, but suggest a role for mechanisms other than EMT.


Assuntos
Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Colo do Útero/metabolismo , Colo do Útero/patologia , Estudos de Coortes , Transição Epitelial-Mesenquimal , Feminino , Humanos , Imuno-Histoquímica , Molécula L1 de Adesão de Célula Nervosa/genética , Prognóstico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Vimentina/genética , Vimentina/metabolismo
13.
Nat Commun ; 11(1): 1943, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327648

RESUMO

Kidney fibrosis is a highly deleterious process and a final manifestation of chronic kidney disease. Alpha-(α)-synuclein (SNCA) is an actin-binding neuronal protein with various functions within the brain; however, its role in other tissues is unknown. Here, we describe the expression of SNCA in renal epithelial cells and demonstrate its decrease in renal tubules of murine and human fibrotic kidneys, as well as its downregulation in renal proximal tubular epithelial cells (RPTECs) after TGF-ß1 treatment. shRNA-mediated knockdown of SNCA in RPTECs results in de novo expression of vimentin and α-SMA, while SNCA overexpression represses TGF-ß1-induced mesenchymal markers. Conditional gene silencing of SNCA in RPTECs leads to an exacerbated tubulointerstitial fibrosis (TIF) in two unrelated in vivo fibrotic models, which is associated with an increased activation of MAPK-p38 and PI3K-Akt pathways. Our study provides an evidence that disruption of SNCA signaling in RPTECs contributes to the pathogenesis of renal TIF by facilitating partial epithelial-to-mesenchymal transition and extracellular matrix accumulation.


Assuntos
Nefropatias/patologia , Rim/patologia , alfa-Sinucleína/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/genética , Vimentina/metabolismo , alfa-Sinucleína/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
Sci Rep ; 10(1): 6657, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313093

RESUMO

As a potential drug/gene delivery system, the ultrasound-targeted microbubble destruction (UTMD) system can be used as a vehicle as well as increasing the permeability of biological barriers to enhance the effect of tumor treatment. However, the effect of UTMD in the tumor EMT process is unknown. In this study, we aimed to investigate the potential and mechanism of UTMD induced oxidative stress in inhibiting EMT of breast cancer. Human breast MDA231 cells were treated with microbubble (MB), ultrasound (US) and UTMD, respectively. The generation of oxidative stress, the levels of miR-200c, ZEB1 and vimentin, and the numbers of migratory cells were evaluated quantitatively and qualitatively by the measurement of intracellular reactive oxygen species (ROS), qRT-PCR, western blot assay, and transwell assay. Then, to evaluate the role of UTMD-induced oxidative stress and miR-200c in the epithelial-mesenchymal transition (EMT) inhibition, the ROS scavenger N-acetyl-L-cysteine (NAC) and miR-200c inhibitor were used before UTMD treatment. We found that UTMD induced oxidative stress, upregulated the expression of miR-200c, downregulated the expression of ZEB1 and vimentin and suppressed the MDA231 cell migration. The addition of NAC and miR-200c inhibitor had an opposite impact on the expression of miR-200c and ZEB1, thus hindered the effects of UTMD on MDA231 cells EMT. In conclusion, UTMD can inhibit the EMT characteristics of MDA231 cells. The mechanism may be related to the regulation of the miR-200c/ZEB1 axis through the generation of ROS induced by UTMD, which may provide a new strategy to prevent the tumor cells EMT under UTMD treatment.


Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Interferente Pequeno/metabolismo , Vimentina/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Acetilcisteína/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos da radiação , Feminino , Depuradores de Radicais Livres/farmacologia , Técnicas de Transferência de Genes , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Microbolhas , Estresse Oxidativo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Ondas Ultrassônicas , Vimentina/antagonistas & inibidores , Vimentina/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/antagonistas & inibidores , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
15.
Genes Cells ; 25(6): 413-426, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32243065

RESUMO

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin, classified as the type III intermediate filament family, maintain the integrity and architecture of various cell types. Recently, we reported their cell surface expression and binding to multivalent N-acetylglucosamine-conjugated polymers. Furthermore, the presence of vimentin on the surface of various cell types including malignant tumor cells and fibroblasts has been demonstrated. Type III intermediate filament proteins are traditionally considered intracellular proteins and do not possess signal peptides for cell membrane recruitment. Therefore, the mechanism of their transport to the cell surface is unclear. In the current study, we aimed to elucidate this mechanism by focusing on the relationship between their multimeric structure and lipid bilayer affinity. Blue native polyacrylamide gel electrophoresis demonstrated that cell surface-expressed type III intermediate filament proteins formed a multimeric mostly including 4-12-mers but not filamentous structure. Moreover, surface plasmon resonance analysis revealed that the multimeric structure of these recombinant proteins had high affinity to lipid bilayers, whereas their filament-like large multimeric structure did not. Our results suggest that type III intermediate filaments are incorporated into the cell membrane through alteration from a filamentous to a multimeric structure.


Assuntos
Membrana Celular/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Bicamadas Lipídicas/metabolismo , Acetilglucosamina/metabolismo , Animais , Linhagem Celular Tumoral , Desmina/genética , Desmina/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Proteínas de Filamentos Intermediários/genética , Cinética , Camundongos , Microscopia de Força Atômica , Periferinas/genética , Periferinas/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Vimentina/genética , Vimentina/metabolismo
16.
Sci Rep ; 10(1): 6127, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273567

RESUMO

It has been suggested that miR-144 is pro-atherosclerotic via effects on reverse cholesterol transportation targeting the ATP binding cassette protein. This study used proteomic analysis to identify additional cardiovascular targets of miR-144, and subsequently examined the role of a newly identified regulator of atherosclerotic burden in miR-144 knockout mice receiving a high fat diet. To identify affected secretory proteins, miR-144 treated endothelial cell culture medium was subjected to proteomic analysis including two-dimensional gel separation, trypsin digestion, and nanospray liquid chromatography coupled to tandem mass spectrometry. We identified 5 gel spots representing 19 proteins that changed consistently across the biological replicates. One of these spots, was identified as vimentin. Atherosclerosis was induced in miR-144 knockout mice by high fat diet and vascular lesions were quantified by Oil Red-O staining of the serial sectioned aortic root and from en-face views of the aortic tree. Unexpectedly, high fat diet induced extensive atherosclerosis in miR-144 knockout mice and was accompanied by severe fatty liver disease compared with wild type littermates. Vimentin levels were reduced by miR-144 and increased by antagomiR-144 in cultured cardiac endothelial cells. Compared with wild type, ablation of the miR-144/451 cluster increased plasma vimentin, while vimentin levels were decreased in control mice injected with synthetic miR-144. Furthermore, increased vimentin expression was prominent in the commissural regions of the aortic root which are highly susceptible to atherosclerotic plaque formation. We conclude that miR-144 maybe a potential regulator of the development of atherosclerosis via changes in vimentin signaling.


Assuntos
MicroRNAs/genética , Placa Aterosclerótica/metabolismo , Vimentina/genética , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Placa Aterosclerótica/genética , Vimentina/metabolismo
17.
Exp Mol Pathol ; 115: 104439, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32283061

RESUMO

Vimentin (VIM) is a mesenchymal marker which is expressed in some cancer types including breast cancer. A long non-coding RNA (lncRNA) has been identified to be transcribed from VIM gene locus and positively regulate expression of VIM. This lncRNA has been named as VIM-antisense 1 (VIM-AS1). Expression of VIM is also regulated by another lncRNA namely AGAP2-antisense RNA 1 (AGAP2-AS1). In the current study, we aimed at identification of the expression pattern of VIM, VIM-AS1, AGAP2 and AGAP2-AS1 in 78 breast cancer samples and their paired adjacent non-cancerous tissues (ANCTs) by means of real time PCR. All mentioned genes were significantly down-regulated in tumoral tissues compared with ANCTs (P values less than 0.000). Relative expression of VIM-AS1 in tumoral tissues versus ANCTs was associated with menopause age (P = .02) in a way that this gene was down-regulated in most of patients whose menopause age was between 40 and 50 years. Moreover, AGAP2-AS1 relative expression was associated with patients' body mass index (P = .03). There were trends toward association between VIM relative expression and tumor size (P = .07) and association between VIM-AS1 relative expression and obesity (P = .06). Expression of VIM was significantly higher in tumoral tissues of patients who had history of hormone replacement therapy compared with those without such history (P = .03). Moreover, expression levels of both VIM and AGAP2-AS1 were lower in patients whose menarche age was between 10 and 12 years old compared with those whose menarche age was between 13 and 15 years old (P values = .01 and 0.04, respectively). Transcript quantities of VIM, VIM-AS1, AGAP2 and AGAP2-AS1 were correlated with each other both in tumoral tissues and in ANCTs. Among four assessed genes, AGAP2 had the best diagnostic power for discrimination of tumoral tissues from ANCTs (AUC value = 0.87). Combination of four genes led to enhancement of AUC value to 0.94. The current study shows the importance of VIM and its associated lncRNAs in breast cancer and potentiates these genes as biomarkers for this malignancy. Moreover, these lncRNAs might be regarded as therapeutic targets in breast cancer.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , RNA Longo não Codificante/genética , Vimentina/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Pessoa de Meia-Idade , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Curva ROC , Vimentina/metabolismo
18.
Oncogene ; 39(18): 3680-3692, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152404

RESUMO

Epithelial-mesenchymal transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified tissue factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (1) vimentin silencing accelerated TF mRNA decay after actinomycin D treatment, reflecting TF mRNA stabilization, (2) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (3) TF 3'-UTR-luciferase reporter vector assays implicated the 3'-UTR of TF mRNA in vimentin-dependent TF regulation, and (4) using different TF 3'UTR-luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.


Assuntos
Neoplasias da Mama/genética , Células Neoplásicas Circulantes/metabolismo , Tromboplastina/genética , Vimentina/genética , Regiões 3' não Traduzidas/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dactinomicina/farmacologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , Metástase Neoplásica , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética
19.
Oncol Rep ; 43(4): 1147-1158, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32020235

RESUMO

Periostin (PN) (also known as osteoblast­specific factor OSF­2) is a protein that in humans is encoded by the POSTN gene and has been correlated with a reduced survival of cholangiocarcinoma (CCA) patients, with the well­known effect of inducing epithelial­to­mesenchymal transition (EMT). The present study investigated the effect of PN, through integrin (ITG)α5ß1, in EMT­mediated CCA aggressiveness. The alterations in EMT­related gene and protein expression were investigated by real­time PCR, western blot analysis and zymogram. The effects of PN on migration and the level of TWIST­2 were assessed in CCA cells with and without siITGα5 transfection. PN was found to induce CCA cell migration and EMT features, including increments in Twist­related protein 2 (TWIST­2), zinc finger protein SNAI1 (SNAIL­1), α-smooth muscle actin (ASMA), vimentin (VIM) and matrix metallopeptidase 9 (MMP­9), and a reduction in cytokeratin 19 (CK­19) together with cytoplasmic translocation of E-cadherin (CDH­1). Additionally, PN markedly induced MMP­9 activity. TWIST­2 was significantly induced in PN­treated CCA cells; this effect was attenuated in the ITGα5ß1­knockdown cells and corresponded to reduced migration of the cancer cells. These results indicated that PN induced CCA migration through ITGα5ß1/TWIST-2­mediated EMT. Moreover, clinical samples from CCA patients showed that higher levels of TWIST­2 were significantly correlated with shorter survival time. In conclusion, the ITGα5ß1­mediated TWIST­2 signaling pathway regulates PN­induced EMT in CCA progression, and TWIST­2 is a prognostic marker of poor survival in CCA patients.


Assuntos
Moléculas de Adesão Celular/genética , Colangiocarcinoma/genética , Integrina alfa5beta1/genética , Proteínas Repressoras/genética , Proteína 1 Relacionada a Twist/genética , Antígenos CD/genética , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Colangiocarcinoma/patologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Transdução de Sinais/genética , Vimentina/genética
20.
Life Sci ; 248: 117469, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32109485

RESUMO

AIMS: Histone deacetylases inhibitors have shown favorable antitumor activity in clinical investigations. In the present study, we assessed the effects of a novel hydroxamic acid-based HDAC inhibitor, SB939, on breast cancer metastasis and tumor growth and characterized the underlying molecular mechanisms. MAIN METHODS: MTS, Wound-healing, and Transwell chamber invasion assays were used to detect the inhibition effects of SB939 on proliferation, migration, and invasion of breast cancer cells. Western blot, cellular immunofluorescence, and EMSA were used to explore the molecular mechanism of SB939 in suppressing breast cancer metastasis. MDA-MB-231 subcutaneous tumor-bearing model of nude mice and the spontaneous metastasis model of breast cancer were both applied to verify in vivo anti-tumor growth and anti-metastatic effects. KEY FINDINGS: Our results demonstrated that SB939 at 0.5-1 µmol/L markedly impaired the chemotactic motility of breast cancer cells. SB939 reversed epithelial-mesenchymal transition (EMT) process, as evidenced by upregulation E-cadherin expression and downregulation expressions of N-cadherin and vimentin through increasing the levels of ac-histone H3 and H4 and drecreasing the expressiongs of HDAC 5 and 4. This cascade inhibition mediated by SB939 was well interpreted by inactivating phosphorylation of STAT3, blocking its DNA-binding activity, and decreasing the expressions of STAT3-dependent target genes, including MMP2 and MMP9. Furhtermore, we found that SB939 significantly inhibited breast cancer metastasis and tumor growth in vivo and showed superior anti-tumor properties compared with SAHA in two breast cancer animal models. SIGNIFICANCE: Our findings indicate that SB939 may be an effective therapeutic option for treating advanced breast cancer.


Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Vimentina/genética , Vimentina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
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