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1.
Methods Mol Biol ; 2578: 161-175, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152286

RESUMO

Peptide microarray provides the ability to miniaturize, parallelize, and automate high-throughput screening substrate specificities of enzymes, profiling of multiple enzyme activities, discovery of disease biomarkers, and development of drugs. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a peptide microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13) activities in the culture medium of four human osteosarcoma (OS) cells and in the progression of OS by using the mouse-bearing xenograft OSs including U-2OS and Saos-2 human. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 with limit of detection downs to 10 pM, 30 pM, 113 pM, 13 pM, 93 pM, and 12 pM, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs is serum closely relevant to the disease progression and type of tumor.


Assuntos
Neoplasias Ósseas , Nanotubos , Osteossarcoma , Óxido de Zinco , Animais , Neoplasias Ósseas/patologia , Fluorescência , Humanos , Metaloproteinase 1 da Matriz , Metaloproteinase 13 da Matriz , Metaloproteinase 2 da Matriz , Metaloproteinase 3 da Matriz , Metaloproteinase 7 da Matriz , Metaloproteinase 9 da Matriz , Camundongos , Osteossarcoma/patologia , Peptídeos , Polímeros
2.
Methods Mol Biol ; 2578: 177-189, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36152287

RESUMO

Peptide array-based in situ fluorescence assay is a reliable and efficient technique for high-throughput profiling and localization of enzyme activity. Here, peptide array is fabricated by spotting five specific MMPs (MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14) peptide substrates containing FAM/Dabcyl fluorescent resonance energy transfer (FRET) pair on the surface of cell monolayers or tissue sections. MMP activities are determined in situ by the fluorescence intensity of stained cells/tissues due to the cellular internalization of hydrolyzed peptide fragments with FAM moieties. Identification of MMP expression patterns of cells, highly sensitive determination of MMP activities in cell monolayer (as low as hundreds of cells per square centimeter), and evaluation of inhibition potencies of six compounds toward five MMPs are achieved by this method. Five MMP activities in the localized parts of 32 thyroid tissues are also well profiled without separation or extraction procedures.


Assuntos
Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Metaloproteinase 13 da Matriz , Metaloproteinase 14 da Matriz , Metaloproteinase 3 da Matriz , Metaloproteinase 7 da Matriz , Fragmentos de Peptídeos/metabolismo , Peptídeos/química
3.
Bioorg Chem ; 130: 106230, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36375352

RESUMO

Colorectal cancer is a type of cancer encountered worldwide and ranks third among all cancer types in terms of incidence. Polyphenols have been shown to have a wide range of biological functions, including a significant impact on cancer start, development, and promotion through regulating many signaling pathways. The aim of this study was to investigate the anticancer effects of isoeugenol based compounds 1, 2 on HT29 colorectal cancer cell line in vitro. MTT test and scratch assay were carried out to determine the effect of these compounds on HT29 cell proliferation and migration respectively. In addition, mRNA expression levels of apoptosis and metastasis-related genes (p53, Bcl2, Bax, Caspase 3, Caspase7, Caspase8, Caspase9, HIF1-α, VEGF, MMP-2, MMP-9) were examined by quantitative real-time PCR. The results indicated that 1 and 2 inhibited HT29 cell proliferation and induced apoptosis by increasing the Bax/Bcl2 ratio and Caspase-9 and Caspase-3 mRNA expression. In conclusion, the results of this study showed that the treatment of these compounds significantly suppressed the mRNA expressions of metastasis-related genes such as Matrix Metalloproteinase-2, Matrix Metalloproteinase-9, Vascular Endothelial Growth Factor and Hypoxia­Inducible Factor 1α.


Assuntos
Neoplasias do Colo , Metaloproteinase 2 da Matriz , Humanos , Metaloproteinase 2 da Matriz/genética , Proteína X Associada a bcl-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Neoplasias do Colo/tratamento farmacológico , Proliferação de Células , Fenóis/farmacologia , RNA Mensageiro
4.
Sci Rep ; 12(1): 18636, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36329090

RESUMO

Periodontitis is a chronic inflammatory disease characterized by the release of matrix metalloproteinases (MMPs) from resident connective tissue cells in tooth-supporting tissues (periodontium). Platelet activation, and the attendant release of pro-inflammatory chemokines such as platelet factor 4 (CXCL4/PF4), are associated with periodontitis although the associated biochemical pathways remain undefined. Here we report that recombinant PF4 is internalized by cultured human gingival fibroblasts (hGFs), resulting in significant (p < 0.05) upregulation in both the production and release of MMP-2 (gelatinase A). This finding was corroborated by elevated circulating levels of MMP-2 (p < 0.05) in PF4-overexpressing transgenic mice, relative to controls. We also determined that PF4 induces the phosphorylation of NF-κB; notably, the suppression of NF-κB signaling by the inhibitor BAY 11-7082 abrogated PF4-induced MMP-2 upregulation. Moreover, the inhibition of surface glycosaminoglycans (GAGs) blocked both PF4 binding and NF-κB phosphorylation. Partial blockade of PF4 binding to the cells was achieved by treatment with either chondroitinase ABC or heparinase III, suggesting that both chondroitin sulfate and heparan sulfate mediate PF4 signaling. These results identify a novel pathway in which PF4 upregulates MMP-2 release from fibroblasts in an NF-κB- and GAG-dependent manner, and further our comprehension of the role of platelet signaling in periodontal tissue homeostasis.


Assuntos
Metaloproteinase 2 da Matriz , Periodontite , Camundongos , Animais , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Fator Plaquetário 4/metabolismo , NF-kappa B/metabolismo , Gengiva , Fibroblastos/metabolismo , Periodontite/metabolismo , Inibidores da Angiogênese/metabolismo , Metaloproteinase 3 da Matriz/metabolismo
5.
Iran J Allergy Asthma Immunol ; 21(5): 549-560, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36341563

RESUMO

It is believed that preformed antibodies are responsible for blood transfusion reactions and transplant rejections. In order to remove a tumor, the tissue must be rejected. On the basis of transfusion reaction and transplantation immunology, we hypothesized that allogeneic serum can inhibit tumor growth when injected intra-tumor. Initially, an in vitro cytotoxicity test was conducted using the C57BL/6 serum (intact or decomplemented) in combination with the BALB/c-originating CT26 cell line.  The CT26 cell line was used to establish a mouse model of colon cancer. When the tumor was palpable, C57BL/6 serum was injected intra-tumor. In addition to tumor size, hypoxia, metastatic capacity, angiogenesis, and metabolic and inflammatory status, we evaluated matrix metalloproteinase-2 (MMP)-2 and 9, vascular endothelial growth factor (VEGF)-A, Cluster of Designation (CD) 31, CD38 and interleukine (IL)-10. An in vitro experiment showed that heat-inactivated C57BL/6 serum had significantly lower cytotoxic effects on BALB/c-derived CT26 cells than intact C57BL/6 serum or BALB/c serum. In vivo experiments revealed that tumor size, HIF-1α, MMP-2, and MMP-9 levels were significantly lower in the experimental group than in the control group. In contrast to control animals, allogeneic serum treatment led to marked reductions in CD31, VEGF-1, CD38, and IL-10 levels. A new approach to serum or plasma therapy and allogeneic vaccines for cancer is intra-tumor injection of allogeneic serum. In light of the ease and availability of allogeneic immunotherapies, allogeneic serum and plasma therapy could potentially be used as an alternative monotherapy or in combination with other therapies.


Assuntos
Neoplasias do Colo , Transplante de Células-Tronco Hematopoéticas , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Neoplasias do Colo/terapia , Neovascularização Patológica/terapia , Camundongos Endogâmicos BALB C , Modelos Animais de Doenças , Imunoterapia
6.
Sci Rep ; 12(1): 20125, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36418859

RESUMO

Vasculogenic mimicry (VM) is closely related to cancer progression and metastasis, contributing to poor prognosis in patients with cancer. Resveratrol (RES) is well known to possess anti-cancer activity. This study explored the new role of RES in VM incidence in human prostate cancer (PCa) PC-3 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, transwell invasion, and three-dimensional culture VM tube formation assays were performed to check the cell viability, invasive ability, and vessel-like networks formation, respectively. VM-related proteins were detected by Western blots. The activity of metalloproteinase-2 (MMP-2) was identified by gelatin zymography. Vascular endothelial cadherin (VE-cadherin) mRNA was assessed by reverse transcriptase-polymerase chain reaction. Nuclear twist expression was observed by immunofluorescence assay. RES reduced serum-induced invasion and VM formation. Serum-induced phosphorylation of erythropoiethin-producing hepatoceullular A2 (EphA2) and the expression of VE-cadherin at the protein and mRNA levels were decreased after RES treatment. RES inhibited serum-induced expression and nuclear localization of twist. Serum-activated AKT signaling pathway, including MMP-2 and laminin subunit 5 gamma-2, was impaired by RES. These results suggested that RES may have an anti-VM effect through suppressing the EphA2/twist-VE-cadherin/AKT signaling cascade in PCa PC-3 cells.


Assuntos
Metaloproteinase 2 da Matriz , Neoplasias da Próstata , Masculino , Humanos , Células PC-3 , Resveratrol/farmacologia , Metaloproteinase 2 da Matriz/genética , Proteínas Proto-Oncogênicas c-akt , Neoplasias da Próstata/tratamento farmacológico , RNA Mensageiro
7.
Artigo em Inglês | MEDLINE | ID: mdl-36361378

RESUMO

BACKGROUND: Matrix Metalloproteinases (MMPs) have been found to have important roles in vascular pathology and may be involved in the occurrence of pre-eclampsia. In this study, the serum levels of MMP-2, -7, -9 in normal pregnant women and pre-eclampsia patients were analyzed to assess their predictive value. METHODS: A total of 1563 pregnant women from Peking University Third Hospital, from February 2021 to October 2021, were enrolled. Serum samples were collected from patients one to three times, during the different trimesters. Among the 102 singleton pre-eclampsia patients, we collected samples from 33 patients in the first trimester (6-13 GW), 33 in the second trimester (14-28 GW), 41 in the third trimester (29-41 GW) and 28 after onset of pre-eclampsia. Samples from each trimester were collected before the onset of pre-eclampsia. Then we selected 35, 37, 43 and 25 samples from 124 healthy pregnant women by matching their age, BMI and gestational weeks, using these as the control groups. Serum levels of MMP-2, -7, -9 were detected by ELISA. The receiver operating characteristic (ROC) curve was used to evaluate their predictive value. RESULTS: Except for the first trimester, MMP-2 and MMP-7 were significantly higher in the pre-eclampsia group (p < 0.5). Additionally, in the pre-eclampsia group, MMP-9 increased significantly in the first trimester and after the onset of pre-eclampsia but decreased significantly in the second and third trimesters (p < 0.5). The ROC curve indicated that MMP-9, MMP-2 and MMP-7 were the best indicators for predicting pre-eclampsia in the first, second and third trimesters, respectively. CONCLUSION: Increased MMP-2 and MMP-7 levels and a decreased MMP-9 level seem to be related to the pathogenesis of pre-eclampsia and are expected to be potential predictors of pre-eclampsia.


Assuntos
Pré-Eclâmpsia , Feminino , Humanos , Gravidez , Biomarcadores , Metaloproteinase 2 da Matriz , Metaloproteinase 7 da Matriz , Metaloproteinase 9 da Matriz , Estudos Prospectivos
8.
Biomed Res Int ; 2022: 2760744, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36408277

RESUMO

Objective: As a highly malignant tumour, malignant rhabdoid tumours of the kidney (MRTK) are prone to metastasis and invasion, while tumour metastasis and invasion are inseparable from matrix metalloproteinases (MMPs) and epithelial-mesenchymal transformation (EMT). Moreover, the key to EMT is remodelling of the cytoskeleton. Therefore, our study is aimed at investigating whether doxycycline hydrochloride (DCH), an inhibitor of MMPs, could reverse EMT in MRTK to exert an antitumour effect by regulating MMPs and the cytoskeleton. Methods: The existence of EMT in MRTK cells was verified by bioinformatics analysis, immunofluorescence, and western blotting (WB). In vitro, the proliferation, migration, and invasion abilities of G401 cells were examined by Cell Counting Kit-8 (CCK-8), scratch, and Transwell assays, respectively. The effect of DCH on tumour growth in tumour-bearing mice was explored in in vivo experiments, and the expression of MMP2 and MMP9 and EMT correlation indexes was measured by immunofluorescence and WB, and the changes in cytoskeletal F-actin and ß-tubulin were measured by fluorescence. Results: The altered extracellular matrix (ECM) composition, EMT, and high expression of MMP2 and MMP9 existed in MRTK. DCH inhibited the proliferation, migration, and invasion of G401 cells in vitro. In vivo, DCH inhibited tumour growth in mice, downregulated the expression of MMP2 and MMP9, and partially reversed EMT. Alternatively, DCH resulted in cytoskeletal rearrangements of G401 cells. Conclusions: DCH, as an MMP inhibitor, is used for the first time in MRTK research, showing good antitumour effects by reversing EMT and potentially providing new therapeutic measures for MRTK treatment.


Assuntos
Neoplasias Renais , Tumor Rabdoide , Camundongos , Animais , Transição Epitelial-Mesenquimal , Doxiciclina/farmacologia , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Invasividade Neoplásica , Movimento Celular , Linhagem Celular Tumoral , Citoesqueleto , Metaloproteinases da Matriz , Aberrações Cromossômicas
9.
Comput Math Methods Med ; 2022: 1643674, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36398072

RESUMO

Background: Transforming growth factor beta-induced protein (TGFBI, encoded by TGFBI gene), is an extracellular matrix protein, widely expressed in variety of tissues. It binds to collagens type I, II, and IV and plays important roles in the interactions of cell with cell, collagen, and matrix. It has been reported to be associated with myocardial fibrosis, and the latter is an important pathophysiologyical basis of atrial fibrillation (AF). However, the mechanism of TGFBI in AF remains unclear. We aimed to detect the potential mechanism of TGFBI in AF via bioinformatics analysis. Methods: The microarray dataset of GSE115574 was examined to detect the genes coexpressed with TGFBI from 14 left atrial tissue samples of AF patients. TGFBI coexpression genes were then screened using the R package. Using online analytical tools, we determined the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, Gene Ontology (GO) annotation, and protein-protein interaction (PPI) network of TGFBI and its coexpression genes. The modules and hub genes of the PPI-network were then identified. Another dataset, GSE79768 was examined to verify the hub genes. DrugBank was used to detect the potential target drugs. Results: In GSE115574 dataset, a total of 1818 coexpression genes (769 positive and 1049 negative) were identified, enriched in 120 biological processes (BP), 38 cellular components (CC), 36 molecular functions (MF), and 39 KEGG pathways. A PPI-network with average 12.2-degree nodes was constructed. The genes clustered in the top module constructed from this network mainly play a role in PI3K-Akt signaling pathway, viral myocarditis, inflammatory bowel disease, and platelet activation. CXCL12, C3, FN1, COL1A2, ACTB, VCAM1, and MMP2 were identified and finally verified as the hub genes, mainly enriched in pathways like leukocyte transendothelial migration, PI3K-Akt signaling pathway, viral myocarditis, rheumatoid arthritis, and platelet activation. Pegcetacoplan, ocriplasmin, and carvedilol were the potential target drugs. Conclusions: We used microdataset to identify the potential functions and mechanisms of the TGFBI and its coexpression genes in AF patients. Our findings suggest that CXCL12, C3, FN1, COL1A2, ACTB, VCAM1, and MMP2 may be the hub genes.


Assuntos
Fibrilação Atrial , Miocardite , Humanos , Fibrilação Atrial/genética , Metaloproteinase 2 da Matriz , Perfilação da Expressão Gênica , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt
10.
BMC Ophthalmol ; 22(1): 428, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357882

RESUMO

BACKGROUND: Intraocular lymphoma (IOL) is a masquerade syndrome that mimics uveitis, making diagnosis difficult. The serum soluble interleukin-2 receptor (sIL-2R), which is cleaved by matrix metalloproteinase (MMP) -2 and MMP-9, has been recognized as a tumor-related biomarker of malignant lymphomas. The aim of this study was to review the reliability of serum and vitreous sIL-2R for distinguishing IOL from uveitis. METHODS: Patients who underwent diagnostic vitrectomy for marked vitreous haze at Hokkaido University Hospital between April 2014 and June 2019 were enrolled. The patients were divided into an IOL group and a uveitis group, according to the pathology of their vitreous samples. The IOL group was further divided at the time of vitrectomy into patients who already had extraocular involvement (IOL with extraocular involvement group) and patients with no evidence of having extraocular involvement (IOL without extraocular involvement group). Serum sIL-2R, and intravitreal sIL-2R, MMP-2, and MMP-9 levels were assessed. RESULTS: Twenty-five eyes of 25 patients, and 15 eyes of 15 patients were included in the IOL group and uveitis group, respectively. The serum sIL-2R levels were significantly lower in the IOL group than in the uveitis group (P < 0.05), and 20.0% and 66.7% in the IOL and the uveitis group showed high sIL-2R value above the normal range. Vitreous sIL-2R tended to be higher in the IOL group than in the uveitis group (P = 0.80). Serum sIL-2R was significantly lower in the IOL without extraocular involvement group than in the IOL with extraocular involvement group (P < 0.05); 5.9% in the IOL without extraocular involvement group and 50.0% in the IOL with extraocular involvement group showed high sIL-2R value above the normal range. Vitreous sIL-2R, MMP-2, and MMP-9 tended to be higher in the IOL with extraocular involvement group than in the IOL without extraocular involvement group (P = 0.30, < 0.05, 0.16). CONCLUSIONS: Serum sIL-2R is often within the normal range in IOL patients. Even if it is within the normal range, the possibility of IOL should be considered. Serum sIL-2R is not a reliable biomarker for IOL, whereas vitreous sIL-2R may be useful for the diagnosis of IOL.


Assuntos
Neoplasias do Sistema Nervoso Central , Neoplasias Oculares , Linfoma Intraocular , Uveíte , Humanos , Metaloproteinase 9 da Matriz , Metaloproteinase 2 da Matriz , Reprodutibilidade dos Testes , Receptores de Interleucina-2 , Biomarcadores Tumorais , Uveíte/diagnóstico , Neoplasias Oculares/diagnóstico
11.
Int J Mol Sci ; 23(21)2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36361610

RESUMO

Cerebrovascular disease is one of the leading causes of disability and death worldwide, and seeking a potential treatment is essential. Trilinolein (TriL) is a natural triacylglycerol presented in several plants. The effects of TriL on cerebrovascular diseases such as cerebral ischemia and carotid stenosis have never been studied. Accordingly, we investigated the protection of TriL on cerebral ischemia/reperfusion (I/R) and vascular smooth muscle cell (VSMC) migration in vivo and in vitro. The cerebral infarction area, the intima to media area (I/M ratio), and proliferating cell nuclear antigen (PCNA)-staining of the carotid artery were measured. Platelet-derived growth factor (PDGF)-BB-stimulated A7r5 cell migration and potential mechanisms of TriL were investigated by wound healing, transwell, and Western blotting. TriL (50, 100, and 200 mg/kg, p.o.) reduced: the cerebral infarction area; neurological deficit; TUNEL-positive apoptosis; intimal hyperplasia; and PCNA-positive cells in rodents. TriL (5, 10, and 20 µM) significantly inhibited PDGF-BB-stimulated A7r5 cell migration and reduced matrix metalloproteinase-2 (MMP-2), Ras, MEK, and p-ERK protein levels in PDGF-BB-stimulated A7r5 cells. TriL is protective in models of I/R-induced brain injury, carotid artery ligation-induced intimal hyperplasia, and VSMC migration both in vivo and in vitro. TriL could be potentially efficacious in preventing cerebral ischemia and cerebrovascular diseases.


Assuntos
Metaloproteinase 2 da Matriz , Músculo Liso Vascular , Apoptose , Becaplermina/farmacologia , Becaplermina/metabolismo , Movimento Celular , Proliferação de Células , Infarto Cerebral/patologia , Hiperplasia/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo
12.
Int J Mol Sci ; 23(21)2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36362072

RESUMO

A kind of hydroxylated polymethoxyflavone (PMFs) existing in the citrus genus, 5-Demethyltangeretin (5-DTAN), has been reported to possess several bioactivities in vitro and in vivo. The aim of this study was to investigate whether acetylation could enhance the anticancer activity and oral bioavailability of 5-DTAN. PC-3 human prostate cancer cells were treated with tangeretin (TAN), 5-DTAN, and 5-acetylated TAN (5-ATAN), and the results showed that the cytotoxic effect 5-ATAN (IC50 value of 5.1 µM) on the cell viability of PC-3 cells was stronger than that of TAN (IC50 value of 17.2 µM) and 5-DTAN (IC50 value of 11.8 µM). Compared to 5-DTAN, 5-ATAN treatment caused a more pronounced DNA ladder, increased the sub-G1 phase population, and induced G2/M phase arrest in the cell cycle of PC-3 cells. We also found that 5-ATAN triggered the activation of caspase-3 and the progression of the intrinsic mitochondrial pathway in PC-3 cells, suggesting the induction of apoptosis. In a cell wound healing test, 5-ATAN dose-dependently reduced the cell migration, and the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was decreased after 48 h of 5-ATAN treatment. Moreover, oral administration of 5-ATAN showed a significantly stronger inhibitory effect on tumor size and tumor weight in tumor-bearing nude mice than those of vehicle or the 5-DTAN group (p < 0.05). Furthermore, pharmacokinetic results showed that single-dose oral administration of 5-ATAN exhibited a higher maximum concentration (Cmax) and area under the curve (AUC) of 5-DTAN in plasma than that of 5-DTAN. More extensive distribution of 5-DTAN to most tissues of mice was also observed in mice treated with 5-ATAN for 7 days. In conclusion, acetylation strongly enhances the anticancer activity and oral bioavailability of 5-DTAN and could be a promising strategy to promote the potential bioactivities of natural products.


Assuntos
Metaloproteinase 2 da Matriz , Triacetonamina-N-Oxil , Masculino , Humanos , Camundongos , Animais , Acetilação , Disponibilidade Biológica , Camundongos Nus , Triacetonamina-N-Oxil/farmacologia , Apoptose , Linhagem Celular Tumoral
13.
BMC Womens Health ; 22(1): 470, 2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36434592

RESUMO

BACKGROUND: Pentamidine has been reported to have many pharmacological effects including anti- protozoal, anti-inflammatory, and anti-tumor activities. The aim of this study is to investigate the potential therapeutic role of Pentamidine and molecular mechanisms of Pentamidine on PI3K/AKT signaling pathway underlying the anti-tumor properties in endometrial cancer. METHODS: Our study was carried out in the central laboratory of Harbin Medical University from 2019 to 2021. Human endometrial cancer cell lines Ishikawa and HEC-1A were treated with Pentamidine. The proliferation ability of cells was investigated by MTS and colony formation assays. The cell cycle distribution was detected by flow cytometry. Cell migration and invasion were analyzed by using the wound healing assay and Transwell assay. Western blotting was performed to measure the levels of AKT, p-AKT, MMP-2, and MMP-9. RESULTS: Our results revealed that treatment of Pentamidine inhibited proliferation, migration and invasion of Ishikawa and HEC-1A endometrial cancer cells. Mechanistic investigation showed that Pentamidine inhibited PI3K/AKT signaling pathway and also reduced the expression of MMP-2 and MMP-9. In addition, co-treatment with PI3K kinase inhibitor LY294002 and Pentamidine leaded to increased repression of cell viability and the protein expression of p-AKT in Ishikawa cells. CONCLUSIONS: Pentamidine suppresses PI3K/AKT signaling pathway, and inhibits proliferation, migration and invasion of EC cells. These findings suggested that Pentamidine might be a potential candidate for treating EC through PI3K/AKT pathway.


Assuntos
Neoplasias do Endométrio , Fosfatidilinositol 3-Quinases , Feminino , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/uso terapêutico , Pentamidina/farmacologia , Pentamidina/uso terapêutico , Proliferação de Células , Transdução de Sinais , Neoplasias do Endométrio/patologia
14.
Artigo em Inglês | MEDLINE | ID: mdl-36430020

RESUMO

Genetic polymorphisms in the matrix metalloproteinases (MMPs) family genes may be associated with cadmium (Cd) levels and its adverse effects. This study investigated the impact of MMP-2 and MMP-9 polymorphisms on Cd levels in 238 residents of a condominium in Rio de Janeiro, Brazil, built over an industrial steel slag waste. Polymorphisms were genotyped using TaqMan validated assays, and the Cd levels were measured in blood (BCd) and urine (UCd) samples by atomic absorption spectrometry. Associations were evaluated by linear correlation coefficients and multiple logistic regression, using odds ratios (OR) and 95% confidence intervals (CI). Mean age was 50 ± 15 years; 58% were female, 69% non-smokers. Mean concentrations for BCd and UCd were 0.70 ± 0.2 µg L-1 and 0.56 ± 0.55 µg L-1, respectively. Smoking status was associated with BCd ≥ 0.70 µg L-1 (OR = 2.9; 95% CI = 1.6-5.9). MMP-9 rs17576 A > G was associated with BCd ≥ 0.70 µg L-1 (OR = 2.11; 95% CI = 1.10-4.05) and UCd ≥ 0.56 µg L-1 (OR = 3.38; 95% CI = 1.82-7.65). Knowing possible individual predisposing factors is essential to understand Cd toxicity, and to improve the monitoring of high-risk populations.


Assuntos
Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Feminino , Humanos , Masculino , Adulto , Pessoa de Meia-Idade , Idoso , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Cádmio/toxicidade , Estudos Transversais , Aço , Brasil , Polimorfismo Genético
15.
Invest Ophthalmol Vis Sci ; 63(12): 27, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36409215

RESUMO

Purpose: The purpose of this study was to investigate the molecular mechanism underlying thyroid-associated ophthalmopathy (TAO) clinical subtypes, to do so, we performed transcriptomic analysis to reveal the expression profile of circular RNAs (circRNAs) in TAO subtypes. Methods: High-throughput RNA-sequencing was performed in six pairs of type I and type II orbital connective tissue samples from patients with TAO. The expression levels of circRNAs and mRNAs in type I and type II samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in another three pairs of type I and type II TAO connective tissue samples. We used bioinformatics predictions to construct a circRNA-microRNA (miRNA)-mRNA network. A protein-protein interaction (PPI) network was constructed based on differential mRNA expression, and the hub genes were determined by the Cytoscape software plugin. Functional and pathway enrichment analyses were performed to elucidate circRNA function. Lentiviral-mediated overexpression of hsa_circ_0007006 and the relationship between hsa_circ_0007006 with COL1A1 and MMP2 were evaluated by Western blotting (WB). Moreover, the differential pathways were assessed by WB. Results: RNA sequencing results predicted a total of 7489 circRNAs and 15,803 mRNAs, with 94 upregulated and 76 downregulated circRNAs and 488 upregulated and 138 downregulated mRNAs. The qRT-PCR analysis of seven dysregulated circRNAs and two major mRNAs validated the RNA-sequencing data. The competing endogenous RNA (ceRNA) network included 7 circRNAs, 23 miRNAs, and 262 mRNAs. Functional analysis revealed several important pathways. Overexpression of hsa_circ_0007006 led to decreased expression levels of COL1A1 and MMP2. Activation of the relaxin signaling pathway differed between the two subtypes. Conclusion: We showed that circRNAs are differentially expressed between type I and type II TAO. We speculate that the hsa_circ_0007006-COL1A1 and MMP2-relaxin signaling pathways are important regulatory axes in the pathogenesis of this disease type and could be considered promising diagnostic and therapeutic targets in the future.


Assuntos
Oftalmopatia de Graves , MicroRNAs , Relaxina , Humanos , RNA Circular/genética , Metaloproteinase 2 da Matriz , Oftalmopatia de Graves/genética , MicroRNAs/genética , RNA Mensageiro/genética , Tecido Conjuntivo
16.
Reprod Biol ; 22(4): 100696, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36327673

RESUMO

Preeclampsia (PE) is a serious obstetric complication, in which trophoblast cell invasion and migration contribute to placental inflammation. In line with the discovery that mRNA prostaglandin endoperoxide synthase 2 (PTGS2) participates in the inflammatory responses in various disorders, our study aims to explore the role of PTGS2 in trophoblast invasion and further in inflammatory response in PE, ultimately providing new therapeutic targets. Bioinformatics analysis was exploited to examine PTGS2 expression in GSE40182 and find inflammatory response-relevant genes in downstream targets of PTGS2. HTR-8/SVneo cells were treated with lipopolysaccharide (LPS) and transfected with short hairpin RNA against PTGS2 (shPTGS2). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorence assays were performed to quantify the expressions of PTGS2 and involved genes (matrix metallopeptidase 2 (MMP-2), tissue inhibitors of metalloproteinase-2 (TIMP-2), p65, p-p65, IκB-α, p-IκB-α, PTGIS, CAV1, AGTR1). The migration and invasion of trophoblasts were detected through wound healing and Transwell assays. We screened out PTGS2 from GSE40182 dataset. LPS promoted cell migration and invasion, the expressions of PTGS2 and MMP-2, and reduced the expression of TIMP-2, while PTGS2 knockdown reversed all above effects of LPS. Activation of nuclear factor kappa-B (NF-κB) pathway was reinforced by LPS which also upregulated CAV1 and AGTR1 levels, and downregulated PTGIS level. Also, the effects of LPS were offset by PTGS2 knockdown. Altogether, PTGS2 silencing reverses the promoting effect of LPS on trophoblast invasion and inflammation in PE, making a breakthrough in the research regarding molecular mechanism of PE.


Assuntos
Pré-Eclâmpsia , Trofoblastos , Humanos , Feminino , Gravidez , Trofoblastos/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , NF-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Placenta/metabolismo , Transdução de Sinais/fisiologia , Movimento Celular/genética , Inflamação/metabolismo
17.
Biomed Res Int ; 2022: 6006981, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36330453

RESUMO

Fyn has been proven to be involved in various cell behaviors and pathophysiological processes. However, the expression and roles of Fyn in trophoblasts remain unclear. Here, we aimed to evaluate the participation of Fyn in trophoblast behavior and function, and the related mechanisms were briefly explored. Fyn expression in the HTR-8/SVneo, JEG-3, and JAR cell lines was evaluated by immunofluorescence, quantitative real-time PCR and western blotting. Fyn expression in human hydatidiform moles was also determined by immunohistochemistry and western blot. To explore the effects of Fyn, HTR-8/SVneo and JEG-3 cells were transfected with Fyn shRNA or overexpression plasmid or treated with the Fyn activity inhibitor SU6656 or ERK1/2 inhibitor U0126. The migration, proliferation, and apoptosis of trophoblast cells were assessed using transwell assays, flow cytometry, and cell counting kit-8 assays, respectively. The production of primary inflammatory cytokines, HLA-G and active matrix metallopeptidase (MMP) 2/9, and the phosphorylation of ERK1/2 and STAT3 were evaluated by ELISA, western blot, or gelatin zymography. The results showed that Fyn was expressed by trophoblast cells, mainly in the cytoplasm and membrane. Fyn expression and activity levels both increased in order from HTR-8/SVneo and JAR to JEG-3. The overexpression of Fyn promoted the proliferation and migration of trophoblast cells and inhibited their apoptosis, while the opposite effects were observed for Fyn knockdown and inhibition. Fyn regulated inflammatory cytokine production in trophoblast cells by promoting TGF-ß and IL-4 secretion while inhibiting IFN-γ and TNF-α secretion. Moreover, HLA-G expression in JEG-3 was positively regulated by Fyn. Fyn also facilitated the expression of active MMP2/9 and the activation of ERK1/2 and STAT3. Besides, it was confirmed that Fyn regulated trophoblast cell activities through ERK1/2 signal pathway by using U0126. Our study first detected the expression of Fyn in trophoblast cells. Fyn played pivotal roles in trophoblast cell behaviors and function, ERK1/2 was one of its targets, and MMP2/9 and STAT3 may also be involved in the regulatory mechanism.


Assuntos
Metaloproteinase 2 da Matriz , Trofoblastos , Gravidez , Feminino , Humanos , Trofoblastos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Linhagem Celular Tumoral , Antígenos HLA-G , Transdução de Sinais , Movimento Celular/genética , Proliferação de Células/genética
18.
Oxid Med Cell Longev ; 2022: 4479905, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36225172

RESUMO

Objective: Examining the role of EBV-miR-BARTs in nasopharyngeal cancer etiology and diagnosis. Method: As the subjects of this study, nasopharyngeal cancer cell lines were chosen and then randomly assigned to one of four groups: the control group, EBV-miR-BART5-3p NC, EBV-miR-BART5-3p mimics, and EBV-miR-BART5-3p inhibitor groups. Utilizing reverse transcription polymerase chain reaction, we determined the levels of gene expression in nasopharyngeal cancer cells that had been treated with EBV-miR-BART5-3p (RT-PCR). The MTT, Transwell, and scratch tests were used to determine the degree to which cells underwent apoptosis, invasion, and migration. The Western blotting method was used in order to examine the protein expression. Result: Compared with normal nasopharyngeal cells, P 0.05 showed that nasopharyngeal cancer cells had greater EBV-miR-BART5-3p expressions and proliferation rates in the control, EBV-miR-BART5-3p NC, and EBV-miR-BART5-3p No statistically significant differences were seen between the mimic groups (P > 0.05); compared with the control group, the proliferation rate of the EBV-miR-BART5-3p inhibitor group was lower with P < 0.05. At a significance threshold of P 0.05, there was no difference in the rates of apoptosis between the control group and the EBV-miR- BART5-3p NC group. Comparing the control group to the EBV-miR-BART5-3p mimics group and the EBV-miR-BART5-3p inhibitors group revealed that the rate of apoptosis was dramatically enhanced in the EBV-miR-BART5-3p inhibitors group but significantly decreased in the control group (P 0.05). When comparing the control group to the EBV-miR-BART5-3p NC group, there was no statistically significant change in the total number of invasive cells (P > 0.05). When comparing the EBV-miR-BART5-3p mimics group to the control group, we found a statistically significant increase in the former and a decrease in the latter (P 0.05). The migration rates of the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimics group did not vary from one another in a way that was statistically significant (P > 0.05). When compared to the control group, the migration rate was considerably (P 0.05) lower in the EBV-miR-BART5-3p inhibitor group. There were no discernible changes identified (P > 0.05) in the levels of Bcl-2 protein expression in the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimic group in a research that compared these three groups. Protein levels of BCL-2 were significantly decreased (P 0.05) in the EBV-miR-BART5-3p inhibitor group, in comparison to the control group. When comparing the control and EBV- miR-BART5-3p NC groups, we found no statistically significant differences in Bax and Caspase-3 protein expression levels (P > 0.05). The protein expressions of Bax and Caspase-3 were statistically significantly greater in the EBV-miR-BART5-3p contrast between the inhibitor and control groups. When comparing the protein expressions of MMP-2 and MMP-9 between the control group, the EBV-miR-BART5-3p NC group, and the EBV-miR-BART5-3p mimics group, there was no statistically significant change (P > 0.05). Protein levels of MMP-2 and MMP-9 were inhibited by EBV-miR-BART5-3p to a greater extent (P 0.05) in the experimental group compared to the control group. Conclusion: The understanding that inhibiting expression of EBV-miR-BART5-3p might reduce the risk of developing nasopharyngeal cancer may help direct clinical treatment for the condition.


Assuntos
MicroRNAs , Neoplasias Nasofaríngeas , Apoptose , Caspase 3/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 4/genética , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , MicroRNAs/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Proteína X Associada a bcl-2/genética
19.
J Biosci ; 472022.
Artigo em Inglês | MEDLINE | ID: mdl-36226369

RESUMO

Pelargonidin has attracted much attention in many cancers. Whether the growth of glioma inhibited by pelargonidin is related to the PI3K/AKT/mTOR pathway is unknown. To examine this, we performed both in vivo and in vitro studies. Twenty-four hours after inoculation, C6 cells received various treatments: CCK8, transwell, flow cytometry, and Western blot were performed. The cell supernatant was collected in order to pretreat HUVEC cells, and tube formation assay was performed. An in situ glioma rat model was constructed and administrated treatment according to its group. After 14 days, the brains were obtained for TUNEL assay, immunohistochemical staining, and Western blot. In vitro studies demonstrated that pelargonidin inhibited the proliferation, migration, and invasion of C6 cells, and promoted apoptosis of C6 cells. Additionally, pelargonidin could inhibit tube formation of HUVECs. We also detected the proteins in the PI3K/AKT/mTOR pathway, and the results indicated that pelargonidin inhibited the phosphorylation of AKT, PI3K, and mTOR, and downregulated VEGF protein. In vivo glioma models were successfully built, and pelargonidin could increase the survival rate of rat, and pathological staining results indicated that pelargonidin increased TUNELpositive cells and decreased micro-vessel density (MVD) through PI3K/AKT/mTOR. Pelargonidin could reduce the relative expression of MMP2, MMP9, N-cadherin, and VEGF proteins, and inhibit the PI3K/AKT/ mTOR pathway. Pelargonidin inhibited the vascularization and metastasis of glioma by blocking the PI3K/ AKT/mTOR pathway.


Assuntos
Glioma , Fosfatidilinositol 3-Quinases , Animais , Antocianinas , Apoptose , Encéfalo/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Glioma/tratamento farmacológico , Glioma/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
20.
Cells ; 11(19)2022 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-36230980

RESUMO

Genome-wide association studies (GWAS) have shown that variants of patched homolog 1 (PTCH1) are associated with lung function abnormalities in the general population. It has also been shown that sonic hedgehog (SHH), an important ligand for PTCH1, is upregulated in the airway epithelium of patients with asthma and is suggested to be involved in airway remodeling. The contribution of hedgehog signaling to airway remodeling and inflammation in asthma is poorly described. To determine the biological role of hedgehog signaling-associated genes in asthma, gene silencing, over-expression, and pharmacologic inhibition studies were conducted after stimulating human airway epithelial cells or not with transforming growth factor ß1 (TGFß1), an important fibrotic mediator in asthmatic airway remodeling that also interacts with SHH pathway. TGFß1 increased hedgehog-signaling-related gene expression including SHH, GLI1 and GLI2. Knockdown of PTCH1 or SMO with siRNA, or use of hedgehog signaling inhibitors, consistently attenuated COL1A1 expression induced by TGFß1 stimulation. In contrast, Ptch1 over-expression augmented TGFß1-induced an increase in COL1A1 and MMP2 gene expression. We also showed an increase in hedgehog-signaling-related gene expression in primary airway epithelial cells from controls and asthmatics at different stages of cellular differentiation. GANT61, an inhibitor of GLI1/2, attenuated TGFß1-induced increase in COL1A1 protein expression in primary airway epithelial cells differentiated in air-liquid interface. Finally, to model airway tissue remodeling in vivo, C57BL/6 wildtype (WT) and Ptch1+/- mice were intranasally challenged with house dust mite (HDM) or phosphate-buffered saline (PBS) control. Ptch1+/- mice showed reduced sub-epithelial collagen expression and serum inflammatory proteins compared to WT mice in response to HDM challenge. In conclusion, TGFß1-induced airway remodeling is partially mediated through the hedgehog signaling pathway via the PTCH1-SMO-GLI axis. The Hedgehog signaling pathway is a promising new potential therapeutic target to alleviate airway tissue remodeling in patients with allergic airways disease.


Assuntos
Remodelação das Vias Aéreas , Asma , Animais , Dermatophagoides pteronyssinus , Estudo de Associação Genômica Ampla , Proteínas Hedgehog/metabolismo , Humanos , Inflamação , Ligantes , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Fosfatos , Pyroglyphidae , RNA Interferente Pequeno , Fator de Crescimento Transformador beta1/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo
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