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1.
Anticancer Res ; 40(5): 2583-2589, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366403

RESUMO

BACKGROUND/AIM: Certain integrins including integrin ß3 facilitate movement and survival of metastatic cancer cells. We examined whether benzoxazine dimer analogue N,N-bis(5-ethyl-2-hydroxybenzyl) methylamine (HM) has anti-metastatic effects. MATERIALS AND METHODS: Cell viability was examined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing and phalloidin-rhodamine assays were performed to evaluate the migration and filopodia formation, respectively. Anoikis resistance was studied by anchorage-independent growth assay. The expression of proteins regulating migration were examined by western blot. RESULTS: HM treatment significantly inhibited growth and survival of detached lung cancer cells as indicated by the reduced colony number and size of anchorage-independent growth analysis. HM inhibited cell migration and suppressed filopodia formation. Protein analysis indicated that the compound dramatically decreased integrin ß3 and its related downstream proteins including active focal adhesion kinase (FAK) and active protein kinase B (AKT); however, integrin ß1 and α5 were found to be unaltered. CONCLUSION: HM shows a potential in targeting integrin ß3 and could be a good candidate for further developed as an anti-metastatic therapy.


Assuntos
Anoikis/efeitos dos fármacos , Antineoplásicos/farmacologia , Benzoxazinas/farmacologia , Movimento Celular/efeitos dos fármacos , Integrina beta3/metabolismo , Antineoplásicos/química , Benzoxazinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares , Modelos Biológicos , Cicatrização/efeitos dos fármacos
2.
PLoS One ; 15(4): e0230507, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255777

RESUMO

The efficiency of in vitro platelet production is considerably low compared with physiological activity due to the lack of pivotal factors that are essential in vivo. We developed an ex vivo platelet production system, introducing human megakaryocytes into an isolated porcine thighbone and culturing in closed circuit. The efficiency of the ex vivo platelet production system was compared to those in vivo and in vitro. CD61+ platelet-like cells were counted by immunostaining and flow cytometry. Results showed that 4.41 ± 0.27 × 103 CD61+ platelet-like cells were produced by 1 × 103 megakaryocytes in the ex vivo system, while 3.80 ± 0.87 × 103 and 0.12 ± 0.02 × 103 were produced in the in vivo and in vitro systems, respectively. Notably, ex vivo and in vitro production systems generated cells that responded well to thrombin stimulation and expressed functional molecules, such as CD62P. Overall, our ex vivo production system was comparable to in vivo production system and produced platelet-like cells that were functionally superior to those produced in vitro. In future, the present ex vivo production system implementing xenogeneic bone marrow would offer a promising alternative for industrial-scale production of platelet-like cells.


Assuntos
Plaquetas/metabolismo , Células da Medula Óssea/citologia , Animais , Antígenos CD34/metabolismo , Plaquetas/citologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Suínos , Trombina/farmacologia
3.
Reprod Biol Endocrinol ; 17(1): 94, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729993

RESUMO

BACKGROUND: Although thyroid dysfunction caused by Hashimoto's thyroiditis (HT) is believed to be related to implantation failure due to the underdevelopment of the receptive uterus, it is unknown whether HT itself, even in the euthyroid state, impairs embryo implantation associated with endometrial receptivity defects. To address whether HT itself can affect endometrial receptivity accompanied by implantation alterations, a euthyroid HT model was established in mice. METHODS: Female NOD mice were immunized twice with thyroglobulin and adjuvant to induce the experimental HT model. Four weeks after the second treatment, the mice were normally mated, and pregnant ones were sacrificed in implantation window for thyroid-related parameter and steroid hormones measurements by electrochemiluminescence immunoassay and enzyme-linked immunosorbent assay and implantation site number calculation by uptake of Chicago Blue dye. In addition, certain morphological features of endometrial receptivity were observed by hematoxylin-eosin staining and scanning electron microscopy, and the expression of other receptivity markers were analyzed by immunohistochemistry, RT-qPCR or Western Blot. RESULTS: HT mice displayed intrathyroidal monocyte infiltration and elevated serum thyroid autoantibody levels without thyroid dysfunction, defined as euthyroid HT in humans. Euthyroid HT resulted in implantation failure, fewer pinopodes, retarded pinopode maturation, and inhibited expression of receptivity markers: estrogen receptor α (ERα), integrin ß3, leukemia inhibitory factor (LIF), and cell adhesion molecule-1 (ICAM-1). Interestingly, despite this compromised endometrial receptivity response, no statistical differences in serum estradiol or progesterone level between groups were found. CONCLUSIONS: These findings are the first to indicate that HT induces a nonreceptive endometrial milieu in the euthyroid state, which may underlie the detrimental effects of HT itself on embryo implantation.


Assuntos
Biomarcadores/metabolismo , Implantação do Embrião , Endométrio/fisiopatologia , Doença de Hashimoto/fisiopatologia , Animais , Endométrio/metabolismo , Endométrio/ultraestrutura , Estradiol/sangue , Feminino , Expressão Gênica , Doença de Hashimoto/sangue , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Masculino , Camundongos Endogâmicos NOD , Microscopia Eletrônica de Varredura , Gravidez , Testosterona/sangue , Tireotropina/sangue
4.
J Exp Clin Cancer Res ; 38(1): 449, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31684995

RESUMO

BACKGROUND: Interleukin-8 (IL-8) plays a vital role in the invasion and metastasis of hepatocellular carcinoma (HCC), and is closely associated with poor prognosis of HCC patients. Integrin αvß3, a member of the integrin family, has been reported to be overexpressed in cancer tissues and mediate the invasion and metastasis of HCC cells. However, the relationship between IL-8 and integrin αvß3 in HCC and the underlying mechanism of IL-8 and integrin αvß3 in the invasion of HCC remains unclear. METHODS: The expression of IL-8, integrin αv and integrin ß3 in HCC cells and tissues was detected by quantitative real-time PCR, Western blot and immunohistochemistry. Transwell assay and Western blot was used to detect the invasiveness, the expression of integrin ß3 and the activation of PI3K/Akt pathway of HCC cells pretreated with IL-8 knockdown or exogenous IL-8. RESULTS: IL-8, integrin αv and integrin ß3 were overexpressed in highly metastatic HCC cell lines compared with low metastatic cell lines. There was a positive correlation between integrin ß3 and IL-8 expression in HCC tissues. IL-8 siRNA transfection reduced HCC cell invasion and the levels of integrin ß3, p-PI3K and p-Akt. IL-8 induced HCC cell invasion and integrin ß3 expression was significantly inhibited by transfection with CXCR1 siRNA or CXCR2 siRNA. When we stimulated HCC cells with exogenous IL-8, cell invasion and the levels of integrin ß3, p-PI3K, and p-Akt increased, which could be effectively reversed by adding PI3K inhibitor LY294002. CONCLUSIONS: Our results suggest that IL-8 promotes integrin ß3 upregulation and the invasion of HCC cells through activation of the PI3K/Akt pathway. The IL-8/CXCR1/CXCR2/PI3K/Akt/integrin ß3 axis may serve as a potential treatment target for patients with HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Integrinas/genética , Integrinas/metabolismo , Neoplasias Hepáticas/genética , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Regulação para Cima
5.
Mar Drugs ; 17(12)2019 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-31771240

RESUMO

Chitosan is sensitive to environmental pH values due to its electric property. This study investigates whether the pH-responsive chitosan assay can provide a simple method to evaluate the aggressive behavior of cancer cells with cell detachment ratio. The epithelial-mesenchymal transition (EMT) is induced with transforming growth factor-ß1 (TGF-ß1) in the human non-small cell lung cancer cell line (A549). EMT-induced cells and untreated cells are cultured on chitosan substrates at pH 6.99 for 24 h, followed by pH 7.65 for 1 h. The cell detachment ratio (CDR) on pH-responsive chitosan rises with an increasing of the TGF-ß1 concentration. The protein array reveals that the expression levels of the α2, α3, α5, ß2, and ß3 integrins are higher in EMT-induced A549 cells than in untreated cells. A further inhibition assay shows that adding ß3 integrin blocking antibodies significantly decreases the CDR of EMT-induced cells from 32.7 ± 5.7% to 17.8 ± 2.1%. The CDR of mesenchymal-type lung cancer cells increases on pH-responsive chitosan through the ß3 integrin. Notably, the CDR can be theoretically predicted according to the individual CDR on the pH-responsive chitosan surface, irrespective of heterogeneous cell mixture. The pH-responsive chitosan assay serves as a simple in vitro model to investigate the aggressive behavior of lung cancer including the heterogeneous cell population.


Assuntos
Bioensaio/métodos , Carcinoma Pulmonar de Células não Pequenas/patologia , Quitosana/química , Neoplasias Pulmonares/patologia , Células A549 , Adesão Celular , Movimento Celular , Transição Epitelial-Mesenquimal , Humanos , Concentração de Íons de Hidrogênio , Integrina beta3/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo
6.
Mediators Inflamm ; 2019: 4567106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31772502

RESUMO

Aim: Aware that Down Syndrome patients present among their clinical characteristics impaired immunity, the aim of this study is to identify the statistically significant differences in inflammation-related gene expression by comparing Down Syndrome patients with Periodontal Disease (DS+PD+) with Down Syndrome patients without Periodontal Disease (DS+PD-), and their relationship with periodontitis as a chronic oral inflammatory clinical feature. Materials and Methods: Case study and controls on eleven Down Syndrome patients (DS+PD+ vs. DS+PD-). RNA was extracted from peripheral blood using a Qiagen PAXgene Blood miRNA Kit when performing an oral examination. A search for candidate genes (92 selected) was undertaken on the total genes obtained using a Scientific GeneChip® Scanner 3000 (Thermo Fisher Scientific) and Clariom S solutions for human, mouse, and rat chips, with more than 20,000 genes annotated for measuring expression levels. Results: Of the 92 inflammation-related genes taken initially, four genes showed a differential expression across both groups with a p value of <0.05 from the data obtained using RNA processing of the patient sample. Said genes were TNFSF13B (p = 0.0448), ITGB2 (p = 0.0033), ANXA3 (p = 0.0479), and ANXA5 (p = 0.016). Conclusions: There are differences in inflammation-related gene expression in Down Syndrome patients when comparing patients who present a state of chronic oral inflammation with patients with negative rates of periodontal disease.


Assuntos
Síndrome de Down/imunologia , Síndrome de Down/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Anexina A3/genética , Anexina A3/metabolismo , Fator Ativador de Células B/genética , Fator Ativador de Células B/metabolismo , Feminino , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Masculino , Doenças Periodontais/imunologia , Doenças Periodontais/metabolismo , Periodontite/imunologia , Periodontite/metabolismo
8.
Mol Vis ; 25: 237-254, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31516309

RESUMO

Purpose: The purpose of this study is to examine the expression profile of genes related to integrin-mediated phagocytosis that are altered by dexamethasone (DEX) and/or αvß3 integrin signaling to gain a better understanding of the molecular basis of phagocytosis and the pathophysiology of glucocorticoid-induced ocular hypertension. Methods: RNA and cell lysates were obtained from human trabecular meshwork (HTM) cells incubated with and without DEX for 4-5 d. The relative level of gene expression was evaluated using the Affymetrix Gene Chip® human gene microarray and quantitative PCR (qPCR). Changes in protein expression were validated using western blots or FACS analyses. The involvement of proteins in phagocytosis was determined using siRNA to knock down the expression of these proteins in an immortalized TM-1 cell line. Changes in the phagocytic activity were measured using pHrodo™-labeled S. aureus bioparticles followed by immunofluorescence microscopy. The effect of αvß3 integrin expression and activity on GULP1 mRNA levels was measured using qPCR in TM-1 cells overexpressing wild type or constitutively active αvß3 integrin. Results: Gene microarrays revealed statistically significant differences (>2 fold) in the expression of seven genes known to be involved in phagocytosis. Three genes (CD36, ABR, and GULP1) were downregulated, while four genes (ITGB3, CHN1, PIK3R1, and MFGE8) were upregulated. The genes were either associated with modulating RAC1 activity (ABR and CHN1) or integrin signaling (CD36, GULP1, ITGB3, PIK3R1, and MFGE8). Another gene, SIRPA, was also downregulated (1.6 fold) but only in one cell strain. qPCR and western blot analyses verified that DEX caused a decrease in SIRPA and GULP1 mRNA and their protein levels, while levels of CHN1 mRNA and its protein were upregulated by DEX. qPCR showed that although ABR mRNA was downregulated compared to non-treated controls after 5 d of treatment with DEX, no change at the protein level was detected. qPCR analysis also revealed that DEX caused an increase in MFGE8 mRNA levels. The levels of CD36 mRNA and protein varied between cell strains treated with DEX and were not statistically different compared to controls. The knockdown of GULP1 and ABR using siRNAs decreased phagocytosis by 40%. Interestingly, GULP1 mRNA levels were also decreased by 60% when αvß3 integrin was overexpressed in TM-1 cells. Conclusion: The DEX-induced inhibition of phagocytosis may be caused by the downregulation of ABR and GULP1 disrupting the αvß5 integrin/RAC1-mediated engulfment pathway. The downregulation of GULP1 by αvß3 integrin further suggests that this integrin may be a negative regulator of phagocytosis by transcriptionally downregulating proteins needed for phagocytosis. In summary, these results represent new insights into the effects of glucocorticoids and integrin signaling on the phagocytic process in the TM.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dexametasona/farmacologia , Proteínas Ativadoras de GTPase/metabolismo , Fagocitose , Proteômica , Malha Trabecular/citologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Linhagem Celular , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Integrina beta3/metabolismo , Ligantes , Masculino , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Fagocitose/efeitos dos fármacos , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de Vitronectina/metabolismo , Staphylococcus aureus/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
9.
Int J Med Sci ; 16(8): 1157-1170, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31523179

RESUMO

Background: Current opinion suggests that expansion of cancer stem cells (CSCs) and activation of pro-tumoral inflammation cascade correlate with cancer progression. Materials and methods: We explored the possible contributions of MRC-5 cancer-associated fibroblasts to the expression profiles of CSC markers and inflammation-associated cell surface molecules. The liver cancer cell lines Bel-7402, SMMC-7721, MHCC-LM3, and HepG2 cultured in conditioned medium (CM) from MRC-5 served as test groups, whereas the liver cancer cell lines cultured in normal medium served as control groups. Results: Flow cytometry revealed that the proportions of CD90+ cells were significantly higher in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, and moderately higher in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, than in controls. The CD90+/CD45- proportions were elevated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, but reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, as compared to controls. Western blotting indicated that Nanog was downregulated in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls; that POU5F1 (OCT4/3) was downregulated in MHCC-LM3-(MRC-5)-CM, but upregulated in Bel-7402-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls, and that CK19 was upregulated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, compared to controls. Proportions of cells expressing Toll-like receptor-1+ (TLR1) and TLR4 were significantly higher in MHCC-LM3-(MRC-5)-CM cells, and moderately higher in HepG2-(MRC-5)-CM cells, than controls. However, the TLR1+ and TLR4+ proportions were lower in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells than controls. Proportions of CD25+ cells were reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, but elevated in MHCC-LM3-(MRC-5)-CM and Bel-7402-(MRC-5)-CM cells, compared to controls. Proportion of CD61+ cells was higher in liver cancer cells cultured in MRC-5-CM than in controls. Proportion of CD14+ cells was lower in HCC cells cultured in MRC-5-CM than in controls. Conclusion: MRC-5 extensively affected the production of CSC markers and inflammation-associated cell surface molecules. Tumor-targeting molecular therapies should consider these findings.


Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Inflamação/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Citometria de Fluxo , Células Hep G2 , Humanos , Integrina beta3/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 1 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Microambiente Tumoral , Ensaio Tumoral de Célula-Tronco
10.
Med Sci Monit ; 25: 5426-5434, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31327865

RESUMO

BACKGROUND The neutrophil inflammatory protein, lipocalin-2 (NGAL), is elevated in certain forms of cardiac hypertrophy and acute heart failure. However, the specific role of NGAL in cardiac hypoxia injury is unclear. This study aimed to elucidate the functional role of NGAL in cardiomyocyte hypoxia injury. MATERIAL AND METHODS Neonatal rat cardiomyocytes were transfected with adenovirus [(Ad-NGAL] to overexpress human-NGAL and then were exposed to hypoxia for 24 h to establish a hypoxia model. Cell inflammation was detected by RT-PCT and ELISA assay. Cell apoptosis was detected by TUNEL assay. Oxidative stress was also detected by commercial kits. RESULTS An increased inflammatory response, apoptosis, and augmented oxidative stress were observed after exposure to hypoxia, while NGAL overexpression in cells increased the expression and release of inflammatory cytokines. NGAL overexpression also increased the number of apoptotic cells and the imbalance of Bax/Bcl-2 protein expression. Moreover, NGAL overexpression increased the levels of reactive oxygen species and oxidase activity, but reduced anti-oxidase activity. Mechanistically, we found that NGAL decreased the expression of integrin ß3, but not the expression of integrin avß3 and avß5, thus inhibiting the downstream protein AKT. When we used the constitutively activated AKT overexpression adenovirus to activate AKT, the deteriorated phenotype by NGAL was counteracted. CONCLUSIONS NGAL can directly affect cardiomyocytes and cause cardiomyocyte deteriorated hypoxia injury through inhibiting integrin ß3 signaling.


Assuntos
Proteínas da Fase Aguda/metabolismo , Hipóxia Celular/fisiologia , Integrina beta3/metabolismo , Lipocalina-2/metabolismo , Lipocalinas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Cardiomegalia/metabolismo , Lipocalina-2/fisiologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/metabolismo
12.
Mol Biol Cell ; 30(14): 1716-1728, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31091172

RESUMO

Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin-Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin's apical localization and trafficking. Targeted silencing of clathrin or the Î³1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum-retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.


Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Clatrina/metabolismo , Endocitose , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Animais , Cães , Proteínas de Fluorescência Verde/metabolismo , Integrina beta3/metabolismo , Células Madin Darby de Rim Canino , Subunidades Proteicas/metabolismo , Proteínas Qa-SNARE/metabolismo
13.
Vox Sang ; 114(4): 330-339, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30900265

RESUMO

BACKGROUND AND OBJECTIVES: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). MATERIALS AND METHODS: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. RESULTS: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. CONCLUSION: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.


Assuntos
Plaquetas/citologia , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Dispositivos Lab-On-A-Chip , Citoesqueleto de Actina/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Citometria de Fluxo , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Microscopia , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Refrigeração
14.
Mol Cancer ; 18(1): 31, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823921

RESUMO

BACKGROUND: Long noncoding RNAs (lncRNAs) have been indicated to play critical roles in cancer development and progression. LncRNA HOXD cluster antisense RNA1 (HOXD-AS1) has recently been found to be dysregulated in several cancers. However, the expression levels, cellular localization, precise function and mechanism of HOXD-AS1 in colorectal carcinoma (CRC) are largely unknown. METHODS: Real-time PCR and in situ hybridization were used to detect the expression of HOXD-AS1 in CRC tissue samples and cell lines. Gain- and loss-of-function experiments were performed to investigate the biological roles of HOXD-AS1 in CRC cell line. RNA pull down, RNA immunoprecipitation and chromatin immunoprecipitation assays were conducted to investigate the mechanisms underlying the functions of HOXD-AS1 in CRC. RESULTS: We observed that HOXD-AS1 was located in the nucleus of CRC cells and that nuclear HOXD-AS1 was downregulated in most CRC specimens and cell lines. Lower levels of nuclear HOXD-AS1 expression were associated with poor outcomes of CRC patients. HOXD-AS1 downregulation enhanced proliferation and migration of CRC cells in vitro and facilitated CRC tumourigenesis and metastasis in vivo. Mechanistic investigations revealed that HOXD-AS1 could suppress HOXD3 transcription by recruiting PRC2 to induce the accumulation of the repressive marker H3K27me3 at the HOXD3 promoter. Subsequently, HOXD3, as a transcriptional activator, promoted Integrin ß3 transcription, thereby activating the MAPK/AKT signalling pathways. CONCLUSION: Our results reveal a previously unrecognized HOXD-AS1-HOXD3-Integrin ß3 regulatory axis involving in epigenetic and transcriptional regulation constitutes to CRC carcinogenesis and progression.


Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Integrina beta3/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Proteínas de Homeodomínio/metabolismo , Humanos , Integrina beta3/metabolismo , Metástase Linfática , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estadiamento de Neoplasias , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Reproduction ; 157(5): 423-430, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30780128

RESUMO

Integrin ß3 (ITGB3), which is the target gene of the miRNA let-7 that can be antagonized by long noncoding RNA (lncRNA) H19, is well known to have a critical role in endometrium receptivity. However, the regulation of ITGB3 in cell-cell or cell-extracellular matrix adhesion and invasion for the maintenance of early pregnancy remains unknown. This study aimed to explore the role of the H19/let-7/ITGB3 axis in regulating trophoblastic spheroid adhesion and in vitro invasion ability using the HTR-8/SVneo cell line and to investigate the expression levels of lncRNA H19 and ITGB3 in human products of conception. The in vitro knockdown of H19 resulted in decreased expression of ITGB3 at the mRNA and protein levels and reduced the adhesion and invasion ability. In the embryonic chorion tissue of spontaneous abortion (SA), the expressions of H19 and ITGB3 at both the mRNA and protein levels decreased. The results of quantitative RT-PCR, Western blot analysis, dual-luciferase report gene and functional miRNA let-7 rescue experiments, adhesion assay and in vitro transwell invasion assay confirmed that H19 regulated trophoblastic spheroid adhesion with endometrial stromal cells through the H19/let-7/ITGB3 axis, thereby providing an improved understanding of the molecular mechanism of SA.


Assuntos
Adesão Celular/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/fisiologia , Esferoides Celulares/fisiologia , Trofoblastos/fisiologia , Aborto Espontâneo/genética , Aborto Espontâneo/metabolismo , Aborto Espontâneo/patologia , Adulto , Ligação Competitiva/genética , Estudos de Casos e Controles , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , MicroRNAs/genética , Gravidez , Trofoblastos/patologia , Adulto Jovem
16.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(1): 227-232, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30738475

RESUMO

OBJECTIVE: To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin ß3 cytoplasmic tail on αⅡbß3-mediated cell function. METHODS: 293T cell lines stably co-expressing human wild type integrin αⅡb and full length ß3 or mutant ß3, including ß3-ΔNITY (ß3 cytoplasmic tail NITY motif deleted), ß3-Δ754 (ß3 cytoplasmic tail TNITYRGT motif deleted) and ß3-Δ759 (ß3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. RESULTS: 293T-αⅡbß3ΔNITY, 293T-αⅡbß3Δ754, 293T-αⅡbß3Δ759 and 293T-αⅡbß3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbß3 cells which expressed full ß3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbß3 cells, the 293T-αⅡbß3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbß3Δ754 cells and 293T-αⅡbß3Δ759 cells failed to adhere or spread on immobilized fibrinogen. CONCLUSION: To the cell spreading function mediated by integrin ß3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different ß3 mutants provide a solid basis for a further analysis of mass spectrometry.


Assuntos
Integrina beta3/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Animais , Células CHO , Adesão Celular , Cricetinae , Cricetulus , Células HEK293 , Humanos , Integrina beta3/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética
17.
Am J Pathol ; 189(4): 900-910, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30653955

RESUMO

Galectin-3 (Gal-3; gene LGALS3) is a member of the ß-galactose-binding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates positively with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the number of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of ß3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.


Assuntos
Galectina 3/fisiologia , Regulação Neoplásica da Expressão Gênica , Integrina beta3/metabolismo , Neoplasias Pulmonares/prevenção & controle , Melanoma Experimental/prevenção & controle , Neovascularização Patológica/prevenção & controle , Animais , Apoptose , Adesão Celular , Proliferação de Células , Humanos , Integrina beta3/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus
18.
Cancer Chemother Pharmacol ; 83(4): 615-624, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30627776

RESUMO

PURPOSE: Cancer cells with stem-like phenotype are frequently proliferative and show high resistance to chemotherapeutic agents. Specific cell markers to identify the cancer stem cells and reverse the drugs resistance are urgent needs in clinic cancer treatment. METHODS: To identify the potential role of integrin ß3 in melanoma stem cells. Flow cytometry and immunofluorescence were performed to detect the expression levels of integrin ß3 and integrin ß3 related signal molecules. qRT-PCR and western blotting were used to detect the signaling pathways induced by integrin ß3. Colony formation analysis and melanoma-bearing mice treatment by chemotherapeutic agents and integrin ß3 inhibitors were used to detect the curative effects. RESULTS: We proved that integrin ß3 could serve as a marker of stem-like cancer cells in melanoma, along with the acquired chemotherapeutic drugs resistance. Furthermore, we observed that the membrane-proximal complex of integrin ß3 with KRAS and Galectin-3 on the surface of melanoma cancer cells could recruit the RalB, resulting in the activation of TBK1. The phosphorylated TBK1 facilitates the activation of NF-κB signaling pathway, leading to the stem-like phenotype and drug resistance development in melanoma. Herein, the combination of cilengitide, an integrin ß3 inhibitor, and chemotherapeutic agents were capable of suppressing the tumor growth and reversing the drug resistance induced by integrin ß3. CONCLUSION: These findings identified integrin ß3 as a driver of melanoma stem-like cells with drug resistance and revealed an innovative strategy in clinic melanoma treatment.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Integrina beta3/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Feminino , Humanos , Integrina beta3/metabolismo , Melanoma/patologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Venenos de Serpentes/administração & dosagem , Venenos de Serpentes/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Theranostics ; 9(1): 265-278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30662566

RESUMO

Epithelial-mesenchymal transition (EMT) is closely associated with the development of drug resistance. Lipid metabolism plays an important role in EMT. This work was to study the cholesterol-lowering drug simvastatin for reversing EMT-associated resistance to chemotherapy via lipid metabolism. METHODS: The combination of simvastatin and paclitaxel was used to overcome the EMT-associated drug resistance. For dual-action on both cancer cells and tumor-associated macrophages (TAM), the tumor microenvironment-activatable multifunctional liposomes were developed for drug codelivery. The liposomes were modified with a hairpin-structured, activatable cell-penetrating peptide that is specifically responsive to the tumor-associated protease legumain. RESULTS: It was revealed simvastatin can disrupt lipid rafts (cholesterol-rich domains) and suppress integrin-ß3 and focal adhesion formation, thus inhibiting FAK signaling pathway and re-sensitizing the drug-resistant cancer cells to paclitaxel. Furthermore, simvastatin was able to re-polarize tumor-associated macrophages (TAM), promoting M2-to-M1 phenotype switch via cholesterol-associated LXR/ABCA1 regulation. The repolarization increased TNF-α, but attenuated TGF-ß, which, in turn, remodeled the tumor microenvironment and suppressed EMT. The liposomal formulation achieved enhanced treatment efficacy. CONCLUSION: This study provides a promising simvastatin-based nanomedicine strategy targeting cholesterol metabolism to reverse EMT and repolarize TAM to treat drug-resistant cancer. The elucidation of the molecular pathways (cholesterol/lipid raft/integrin ß3/FAK and cholesterol-associated LXR/ABCA1 regulation) for anti-EMT and the new application of simvastatin should be of clinical significance.


Assuntos
Antineoplásicos/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/imunologia , Paclitaxel/metabolismo , Sinvastatina/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Portadores de Fármacos/metabolismo , Adesões Focais/efeitos dos fármacos , Xenoenxertos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Integrina beta3/metabolismo , Lipossomos/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Paclitaxel/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/administração & dosagem , Resultado do Tratamento
20.
Clin Transl Oncol ; 21(8): 1052-1060, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30632010

RESUMO

BACKGROUND: Integrins are highly attractive targets in oncology due to their involvement in angiogenesis in a wide spectrum of cancer entities. Among several integrin inhibitors, cilengitide is suggested to be one of the most promising inhibitors. However, little is known about the cellular processes induced during cilengitide chemotherapy in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: For the current study, 3 HNSCC cell lines, SCC4, SCC15 and SCC25; and 3 primary culture cells, TU53, TU57, and TU63 were used. CD90, cytokeratin, and vimentin were stained immunohistochemically to identify the biological characteristics of these cell lines and primary culture cells and the cytostatic effect of cilengitide was evaluated. Quantitative polymerase chain reaction (qPCR) arrays were applied to evaluate target protein genes ITGAV, ITGB3, and ITGB5 of integrin αvß3 and αvß5 at respective concentrations of 50 and 100 µM cilengitide for 72 h. RESULTS: Cilengitide has significantly inhibited the proliferation of HNSCC cells in a dose-dependent way. At the same concentration, cilengitide suppressed the proliferation of primary culture cells even more strongly than it did that of cell lines, suggesting that primary culture cells retain more of their internal biological characteristics than do cell lines. qPCR assay detected downregulation of ITGAV, ITGB3, and ITGB5 gene expression after exposure to 50 µM of cilengitide. However, after exposure to 100-µM cilengitide, expression of these genes significantly increased both in cell lines and primary culture cells. CONCLUSIONS: RGD-containing small-molecule synthetic peptides might be considered in tumor chemotherapy in the near future. The different reactions of primary culture cells and cell lines demonstrated that individualized chemotherapy plans may be a feasible option. However, research on the role of cilengitide in HNSCC therapy is still in its early stages, and further investigations are required.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/patologia , Cadeias beta de Integrinas/química , Integrina beta3/química , Venenos de Serpentes/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Apoptose/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Células Tumorais Cultivadas
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