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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.
Contrast Media Mol Imaging ; 2022: 7445042, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36072638

RESUMO

The effect of the TGF-ß pathway-based pituitary tumor of rats on the GH3 cell line after intervention with different concentrations of troglitazone (TGZ) is explored. The CH3 cell line of 24 clean male SD rats with pituitary adenoma is selected. The cells are divided into a blank contrast set and an experimental set. The experimental set is divided into different TGZ concentration sets, including 1 × 10-3 TGZ set, 1 × 10-4 TGZ set, and 1 × 10-5 TGZ set. The cell proliferation is detected by the CCK-8 method, the protein expressions of CD147, TGF-ß1, and MMP-9 are detected by the western blot method, and the relative mRNA expressions of CD147, TGF-ß1, and MMP-9 are detected by the qRT-PCR method. The expression levels of CD147, TGF-ß1, and MMP-9 in CH3 cells of pituitary adenoma rats are notoriously lower, while the expression of CD147, TGF-31, and MMP-9 could be reduced by TGZ acting on the GH3 cell line. The specific mechanism of action of this effect on the invasive ability of GH3 cell lines is multifaceted, suggesting that peroxisome proliferator activator-receptor (PPAR-γ) agonists have good clinical application prospects in tumor therapy and can provide new targets and approaches for tumor drug therapy.


Assuntos
Neoplasias Hipofisárias , Tiazolidinedionas , Animais , Linhagem Celular , Cromanos/farmacologia , Masculino , Metaloproteinase 9 da Matriz , Neoplasias Hipofisárias/genética , Ratos , Ratos Sprague-Dawley , Tiazolidinedionas/farmacologia , Fator de Crescimento Transformador beta , Fator de Crescimento Transformador beta1 , Troglitazona
4.
Molecules ; 27(17)2022 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-36080186

RESUMO

A PEGylated niosomal formulation of cyclophosphamide (Nio-Cyclo-PEG) was prepared using a central composite design and characterized in terms of drug loading, size distribution, and average size. The stability of formulations was also studied at different conditions. In vitro cytotoxicity of drug delivery formulations was assessed on gastric cancer cells using MTT assay. The mechanism of cytotoxicity was studied at the transcriptional level by real-time PCR on Caspase3, Caspase9, CyclinD, CyclinE, MMP-2, and MMP-9 genes, while apoptosis was investigated with flow cytometry. The anti-metastatic property was evaluated using the scratch method. Propidium iodide staining was used to study the cell cycle. The results indicated that the as-designed nanocarrier exhibited a controlled drug release pattern with improved nanoparticle stability. It was found that the living cancer cells treated with Nio-Cyclo-PEG showed a significant decrease in number when compared with the niosomal carrier without PEG (Nio-Cyclo) and free drug (Cyclo). Moreover, the drug-loaded nanocarrier induced planned death (apoptosis) in the cancer cells through the regulation of Caspase3, Caspase9, CyclinD, CyclinE, MMP-9, and MMP-2 gene expression, indicating that the Nio-Cyclo-PEG formulation could significantly inhibit the cell cycle at the sub G1 phase as well as prevent the migration of cancer cells. In conclusion, Nio-Cyclo-PEG as developed in this study could serve as an active-targeting drug delivery nanocarriers for gastric cancer therapy with high efficacy and minimal side effects on healthy tissues/cells.


Assuntos
Nanopartículas , Neoplasias Gástricas , Ciclofosfamida , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz , Polietilenoglicóis , Neoplasias Gástricas/tratamento farmacológico
5.
Dis Markers ; 2022: 5178122, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36061350

RESUMO

Objective: This study is aimed at investigating the molecular mechanism of lncRNA GAS5-induced cell apoptosis in acute myeloid leukemia (AML) by targeting Nrf2. Methods: The RNA interfering technique was utilized to silence THP-1 in AML cell line, and lncRNA GAS5 expression in cell line was determined by real-time PCR. EdU experiment and flow cytometry were used to detect the apoptosis and proliferation ability of cells in different groups. PD-L1, STAT3, AKT, and MMP9 expressions were determined by Western blot. Results: The si-RNA significantly inhibited the expression of lncRNA GAS5 in THP-1 cells. Compared with the si-NC group, the difference in cell apoptosis between lncRNA GAS5 and Nrf2 groups was significant (P < 0.05). Compared with the lncRNA GAS5 group, the number of apoptotic cells in the lncRNA GAS5+Nrf2 group significantly reduced (P < 0.05). Compared with the si-NC group, the differences in the levels of four proteins between lncRNA GAS5 and Nrf2 groups were significant (P < 0.05). In lncRNA GAS5+Nrf2 and lncRNA GAS5 groups, PD-L1 expression increased, while the expression of STAT3, AKT, and MMP9 decreased. Conclusion: In AML cells, lncRNA GAS5 with Nrf2 could regulate the proliferation and apoptosis of AML cells. lncRNA GAS5 inhibited Nrf2 expression, regulated cell apoptosis and proliferation, and further inhibited the progression of AML disease.


Assuntos
Leucemia Mieloide Aguda , Fator 2 Relacionado a NF-E2 , RNA Longo não Codificante , Apoptose , Antígeno B7-H1 , Humanos , Leucemia Mieloide Aguda/genética , Metaloproteinase 9 da Matriz/genética , Fator 2 Relacionado a NF-E2/genética , Proteínas Proto-Oncogênicas c-akt , RNA Longo não Codificante/genética
6.
Kardiologiia ; 62(8): 11-18, 2022 Aug 30.
Artigo em Russo, Inglês | MEDLINE | ID: mdl-36066982

RESUMO

Aim      To study the incidence and clinical and pathophysiological features of diastolic dysfunction (DD) and chronic heart failure with preserved ejection fraction (HFpEF) in patients with resistant arterial hypertension (RAH) associated with type 2 diabetes mellitus (DM).Material and methods  A cross-sectional study that included 36 patients with RAH associated with type 2 DM (mean age, 61.4±6.4 years; 14 men) was performed. Measurement of office and 24-h blood pressure (BP), standard echocardiography with assessment of diastolic function (DF) and ventricular-arterial coupling, doppler ultrasound imaging of renal blood flow, and laboratory tests (blood glucose, glycated hemoglobin, blood creatinine, tumor necrosis factor α (TNF-α), brain natriuretic peptide (BNP), type 2 and type 9 matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of MMP 1 (TIMP-1), 24-h urine protein test, and 24-h urine volume test were performed for all patients. HFpEF was diagnosed according to criteria of the American Society of Echocardiography and the European Society of Cardiology 2019, and the Russian Clinical Guidelines on Diagnosis and Treatment of CHF 2017 and 2020.Results All patients had DD. Incidence of HFpEF detection according to the Russian Guidelines 2017 was 100%; according to the Russian Guidelines 2020, that included a required increase in BNP, and according to the criteria of the European Guidelines 2019, this incidence was 89 %. In 55.6 % of patients, DD corresponded to grade 2 (pseudonormal type). According to the correlation analysis, the DF impairment was associated with increases in pulse BP, myocardial mass, arterial and left ventricular elastance (arterial wall and left ventricular elasticity), basal glycemia and DM duration, MMP-2 level, proteinuria, blood creatinine, renal vascular resistance, and also with decreases in 24-h urine volume, MMP-9, TIMP-1, and TIMP-1/MMP-2. Significance of the relations of mean E / e' ratio with nighttime pulse BP, MMP-9, and 24-h urine volume were confirmed by results of multiple linear regression analysis. Increased myocardial and vascular wall stiffness, concentrations of MMP-2 and TNF-α and reduced 24-h urine volume were associated with progressive impairment of DF.Conclusion      The combination of RAH and DM-2 is characterized by an extremely high incidence of DD that determines a great prevalence of HFpEF. The development and progression of DD in such patients are closely related with a complex of metabolic, proinflammatory and profibrotic biomarkers, increased vascular wall stiffness, pronounced left ventricular hypertrophy, and with structural and functional alterations in kidneys.


Assuntos
Diabetes Mellitus Tipo 2 , Insuficiência Cardíaca , Hipertensão , Idoso , Creatinina , Estudos Transversais , Diabetes Mellitus Tipo 2/complicações , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/etiologia , Humanos , Hipertensão/complicações , Hipertensão/diagnóstico , Hipertensão/epidemiologia , Masculino , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico , Volume Sistólico , Inibidor Tecidual de Metaloproteinase-1 , Fator de Necrose Tumoral alfa
7.
Eur Rev Med Pharmacol Sci ; 26(16): 5710-5717, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36066144

RESUMO

OBJECTIVE: The aim of this study was to investigate the correlations of cluster of differentiation 147 (CD147) with plaque stability of carotid atherosclerosis (AS), degree of stenosis, inflammatory factors, matrix metalloproteinase-9 (MMP-9) expression and vascular endothelial function in patients with cerebral infarction. PATIENTS AND METHODS: A total of 50 patients diagnosed with cerebral infarction (cerebral infarction group), 70 patients diagnosed with AS plaque (plaque group, with no infarction but plaques only) and 30 healthy people receiving physical examination (control group) in our hospital from March 2018 to July 2019 were collected. The levels of biochemical indexes, CD147, MMP-9, vascular endothelial function indexes [endothelin-1 (ET-1) and C-reactive protein (CRP)] and inflammatory factors [interleukin-10 (IL-10), IL-16 and tumor necrosis factor-alpha (TNF-α)] in the blood of each group of patients were detected via radioimmunoassay and enzyme-linked immunosorbent assay (ELISA). Moreover, ultrasonic examination and Gensini score system were applied to score the degree of carotid stenosis in cerebral infarction group. Finally, the differences in various parameters were compared among the three groups, and the correlations of CD147 with different indexes were evaluated using Spearman method. RESULTS: Compared with those in control group, the levels of CD147, MMP-9, hemoglobin, platelets, total cholesterol, triglyceride, low-density lipoprotein (LDL), high-density lipoprotein (HDL), apolipoprotein A, apolipoprotein B, IL-10, IL-13 and TNF-α in the blood were remarkably elevated in cerebral infarction group and plaque group (p<0.05). Cerebral infarction group had notably higher levels of CD147, hemoglobin, triglyceride, apolipoprotein B, IL-10, IL-13 and TNF-α in the blood than plaque group (p<0.05). The plaque score was markedly higher in cerebral infarction group than that in plaque group [(3.27±2.86) points vs. (0.93±1.44) points] (p<0.05). In comparison with control group, plaque group and cerebral infarction group exhibited evidently raised levels of blood ET-1 and CRP (p<0.05). The serum CD147 level was significantly associated with MMP-9 (p=0.003, r=0.616), Gensini score (p=0.006, r=0.656), plaque score (p=0.027, r=0.396), IL-10 (p=0.004, r=0.603), TNF-α (p=0.001, r=0.746) and CRP (p=0.037, r=0.450) in cerebral infarction group. CONCLUSIONS: CD147 level is prominently increased in carotid AS and closely related to inflammatory responses, and CD147 may become a new reference for the prediction and treatment of AS and cerebral infarction.


Assuntos
Aterosclerose , Doenças das Artérias Carótidas , Placa Aterosclerótica , Proteína C-Reativa , Infarto Cerebral , Humanos , Interleucina-10 , Interleucina-13 , Metaloproteinase 9 da Matriz/metabolismo , Placa Aterosclerótica/patologia , Triglicerídeos , Fator de Necrose Tumoral alfa
8.
BMC Neurol ; 22(1): 359, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127663

RESUMO

In the present study, we explored multiple plasma factors to predict the outcomes of patients with AIS after IVT. Fifty AIS patients who received IVT with alteplase were recruited and divided into two groups according to their NIHSS scores. Serum from all subjects was collected to quantitatively analyze the levels of different plasma factors, IL-6, MMP-9, ADAMTS13, TNC, GSN and TRX, using Luminex assays or ELISA measurements. Compared with the levels assessed at the onset of AIS, the levels of MMP-9 (P < 0.001), ADAMTS13 (P < 0.001), and TRX (P < 0.001) significantly decreased after IVT. The level of IL-6 was significantly increased in the NIHSS > 5 group at admission (P < 0.001) compared to the NIHSS ≤ 5 group. AIS patients with a poor prognosis had lower levels of ADAMTS13 at 72 h post-IVT compared with patients with a good prognosis (P = 0.021). IL-6 also was notably higher in the poor outcome group (P = 0.012). After adjusting for confounders, ADAMTS13 at 72 h post-IVT was an independent protective factor for prognosis in AIS patients with an adjusted OR of 0.07 (P = 0.049), whereas IL-6 was an independent predictor of risk for AIS patients with an adjusted OR of 1.152 (P = 0.028). IVT decreased MMP-9, ADAMTS13, and TRX levels in the plasma of AIS patients. Patients with a NIHSS score of less than 5 exhibited lower IL-6 levels, indicating that increased levels of IL-6 correlated with AIS severity after IVT. Therefore, IL-6 and ADAMTS13 might be useful plasma markers to predict the prognosis in AIS patients at 90-days after IVT.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Isquemia Encefálica/tratamento farmacológico , Fibrinolíticos/uso terapêutico , Humanos , Interleucina-6 , Metaloproteinase 9 da Matriz , Prognóstico , Acidente Vascular Cerebral/terapia , Ativador de Plasminogênio Tecidual/uso terapêutico
9.
Int J Mol Sci ; 23(17)2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36077255

RESUMO

Rosacea is a chronic inflammatory skin disease whose prevalence rates remain unknown in Chile. Laboratory benchmark testing for this disease is not useful, therefore, we aimed to evaluate the gingival crevicular fluid (GCF) levels of extracellular metalloproteinases (MMP)-2 and MMP-9 as novel rosacea biomarkers. We designed a cross-sectional study with a control group. Participants were systemically healthy adults (n = 20) and persons with rosacea (n = 18). We performed a periodontal evaluation and collected gingival crevicular fluid to measure MMP-2 and MMP-9 levels. Analysis showed mean and standard deviation of MMP-9 concentrations in the GCF for patients with rosacea was 764.52 ± 569.83 pg/mL; for healthy patients, it was 260.69 ± 170.43 pg/mL (p < 0.05). The diagnosis of rosacea was responsible for the levels of MMP-9 in the GCF (p < 0.05), as opposed to periodontitis, smoking, and age (p > 0.05). The Area under ROC for MMP-9 was 0.869 (95%, C.I: 0.719-0.956), with a sensitivity of 72.22% and specificity of 81.58% for the diagnosis of rosacea. We conclude that the quantification of MMP-9 in the GCF could be used as a biomarker of rosacea. Also, rosacea was responsible for increasing the levels of MMP-9 in the GCF independent of periodontal status.


Assuntos
Líquido do Sulco Gengival , Rosácea , Adulto , Biomarcadores/análise , Chile , Estudos Transversais , Humanos , Metaloproteinase 9 da Matriz , Rosácea/diagnóstico
10.
Wiad Lek ; 75(8 pt 2): 1975-1978, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36129081

RESUMO

OBJECTIVE: The aim: The aim of the study was to determine the content of metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) in the skin of rats of different ages after closure of the wound bed. PATIENTS AND METHODS: Materials and methods: The studies were performed on 40 white nonlinear male rats, 20 of which were 3 months old and 20 - 12 months. In each group 10 rats were control and in 10 others facelift operations were performed and cut wounds on the anterior abdominal wall were simulated. On the day of complete healing, the animals were killed, and the skin was cut in the areas of the former wound bed. In control rats, the skin was excised in the same places. The content of MMPs was determined in the skin by enzyme-linked immunosorbent assay. RESULTS: Results: In rats aged 3 months after re-epithelialization of the wound bed, the content of MMP-2 was 17,1% higher compared to control rats but the level of MMP-9 didn't change. In control rats aged 12 months, the levels of MMP-2 and MMP-9 in the skin were 22,9% and 34,4% lower compared to control rats at 3 months of age. In rats 12 months of age after re-epithelialization of the wound bed, the content of MMP-2 and MMP-9 were 92,6% and 102,5% higher compared to control rats. CONCLUSION: Conclusions: We suggested that the violation of homeostasis between MMPs in rats 12 months of age disrupts wound healing and promotes the formation of pathological scars.


Assuntos
Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Animais , Masculino , Metaloproteinases da Matriz , Pele/patologia , Cicatrização
11.
Genet Res (Camb) ; 2022: 2823356, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36118275

RESUMO

Matrix metalloproteinase (MMP)-2 and MMP-9 are a family of Zn2+ and Ca2+-dependent gelatinase MMPs that regulate muscle development and disease treatment, and they are highly conservative during biological evolution. Despite increasing knowledge of MMP genes, their evolutionary mechanism for functional adaption remains unclear. Moreover, analysis of codon usage bias (CUB) is reliable to understand evolutionary associations. However, the distribution of CUB of MMP-2 and MMP-9 genes in mammals has not been revealed clearly. Multiple analytical software was used to study the genetic evolution, phylogeny, and codon usage pattern of these two genes in seven species of mammals. Results showed that the MMP-2 and MMP-9 genes have CUB. By comparing the content of synonymous codon bases amongst seven mammals, we found that MMP-2 and MMP-9 were low-expression genes in mammals with high codon conservation, and their third codon preferred the G/C base. RSCU analysis revealed that these two genes preferred codons encoding delicious amino acids. Analysing what factors influence CUB showed that the third base distributors of these two genes were C/A and C/T, and GC3S had a wide distribution range on the ENC plot reference curve under no selection or mutational pressure. Thus, mutational pressure is an important factor in CUB. This study revealed the usage characteristics of the MMP-2 and MMP-9 gene codons in different mammals and provided basic data for further study towards enhancing meat flavour, treating muscle disease, and optimizing codons.


Assuntos
Uso do Códon , Metaloproteinase 2 da Matriz , Aminoácidos/genética , Animais , Análise por Conglomerados , Códon/genética , Mamíferos/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética
12.
Dis Markers ; 2022: 5085183, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36118675

RESUMO

Background: Chronic actinic dermatitis (CAD) is an abnormally proliferating photoallergic skin disease. Dysregulated inflammation and oxidative stress are the immediate factors in the abnormal proliferation of keratinocytes. This study aimed to investigate the effect of curcumin on the aberrant proliferation of keratinocytes in an in vitro (actinic dermatitis) AD model and the possible molecular mechanisms. Methods: The keratinocytes were irradiated with ultraviolet (UV) to construct an in vitro AD model and then processed with different concentrations of curcumin. Cell viability, oxidative stress markers (SOD, GSH-PX, and MDA), activated oxygen species (ROS), and inflammation markers (IL-1ß, IL-6, IL-18, and TNFα) were determined, respectively. Western blot was applied to assay the profiles of apoptosis-related proteins (Bax, Bcl-xL, Caspase3, Caspase8, and Caspase9), oxidative stress proteins (Keap1, Nrf2, HO-1, COX2, and iNOS), and inflammatory proteins (NF-κB, MMP1, and MMP9) and SPAG5/FOXM1. Functionally, SPAG5 or FOXM1 overexpression and knockdown models were constructed in keratinocytes to characterize their influence on UV irradiation-mediated keratinocyte dysfunction. Results: Curcumin weakened UV-mediated inflammation, proliferation, and oxidative stress and impaired apoptosis in keratinocytes. UV boosted SPAG5/FOXM1 expression in cells, while curcumin concentration-dependently retarded SPAG5/FOXM1 expression. Overexpression of SPAG5/FOXM1 fostered UV-mediated inflammation, proliferation, oxidative stress, and intensified apoptosis, whereas curcumin mostly reversed the SPAG5/FOXM1-mediated effects. In addition, knocking down SPAG5/FOXM1 ameliorated UV-mediated keratinocyte dysfunction, whereas curcumin failed to exert further protective effects in cells with knockdown of SPAG5/FOXM1. Conclusion: Curcumin modulated proliferation, inflammation, oxidative stress, and apoptosis of keratinocytes by restraining the SPAG5/FOXM1 axis.


Assuntos
Curcumina , Transtornos de Fotossensibilidade , Proteínas de Ciclo Celular , Proliferação de Células , Curcumina/metabolismo , Curcumina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/farmacologia , Proteína Forkhead Box M1 , Humanos , Inflamação/metabolismo , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Queratinócitos/metabolismo , Metaloproteinase 1 da Matriz , Metaloproteinase 9 da Matriz/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Oxigênio/farmacologia , Transtornos de Fotossensibilidade/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologia
13.
Biomed Res Int ; 2022: 1840541, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36158893

RESUMO

In this study, we have examined the anticancer effects of SH005S7 on MET-amplified and (HCC827GR) NSCLC cells and their primary HCC827 cells. In vitro, first of all, cell viability and colony formation assay confirmed the growth inhibitory effects of SH005S7 on both cells. Second, SH005S7 inactivated EGFR-related multiple cell signaling, which was associated with a marked decrease in the constitutive phosphorylation of EGFR, HER3, MET, AKT, and ERK. Third, SH005S7 attenuated the anchorage-independent cell growth. Fourth, SH005S7 blocked invasive and metastatic capability by downregulation of mesenchymal markers-vimentin, snail, and MMP-9. Fifth, BrdU assay confirmed the cell cycle arrest of SH005S7 on these cells. When administered orally to nude mice xenografically transplanted human NSCLC, SH005S7 inhibited the growth of tumor and did not cause hepatotoxicity and nephrotoxicity in animals. Immunohistochemical and Western blot analyses of tissue showed that the suppression of growth correlated with inhibition of proliferation (Ki-67, PCNA), invasiveness (vimentin, snail), and angiogenesis (CD31) marker and decrement in the constitutive and phosphorylation of EGFR, HER3, MET, AKT, and ERK. Additionally, SH005S7 had immune stimulatory effects by TNF-α cytokine release on macrophage, without cell cytotoxicity. Overall, our results suggest that SH005S7 can inhibit the growth of MET-amplified and gefitinib-resistant NSCLC cells through the suppression of EGFR-related multiple targets linked to overcome gefitinib resistance.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Apoptose , Bromodesoxiuridina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Humanos , Antígeno Ki-67 , Neoplasias Pulmonares/patologia , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Nus , Antígeno Nuclear de Célula em Proliferação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-met/metabolismo , Quinazolinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Vimentina
14.
Cell Death Dis ; 13(9): 814, 2022 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-36138026

RESUMO

Tyrosine kinase inhibitors (TKIs) are the most commonly used targeted therapeutics in clear-cell renal cell carcinoma (ccRCC); however, drug resistance limits their utility and can lead to tumor "flare-up" and progression. In this study, we show that RCC resistance to sunitinib and sorafenib involves different mechanisms and leads to increased malignancy. Sunitinib decreased tumor growth and cell motility along with increased E-cadherin expression and secretion of the proangiogenic cytokines IL6 and IL8, which activated senescence in ccRCC cells and led to VE-cadherin phosphorylation, enhancing tumor angiogenesis. Sorafenib resistance increased the levels of mesenchymal markers and the secretion of MMP9, which cleaved VE-cadherin and disrupted endothelial cell integrity. Both sunitinib resistance and sorafenib resistance led to activation of the c-Met receptor IRAK1 and downregulation of the tumor suppressor MCPIP1, resulting in an increase in the metastasis of resistant cells, possibly due in part to enhanced vascularization of ccRCC. MCPIP1 overexpression partially overcame resistance to these drugs by decreasing micrometastasis and decreasing the expression of factors involved in tumorigenesis. In tumor samples from ccRCC patients, we observed a significant increase in the level of the c-Met receptor, IRAK1 and a decrease in MCPIP1 with respect to normal kidney tissue. Our results indicate separate novel mechanisms for sunitinib and sorafenib resistance, which both lead to MCPIP1 inhibition and ccRCC progression. The presented study suggests caution in the treatment of RCC with TKIs, which may lead to the unintended outcome of tumor progression.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Caderinas/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Rim/patologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neovascularização Patológica/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico
15.
J Immunol Res ; 2022: 7465353, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36132983

RESUMO

Purpose: To investigate the function of C/EBPα in the development of aortic dissection (AD) and the underlying mechanism. Methods: Aortic vascular smooth muscle cells (VSMCs) were isolated, cultured, and identified from AD rats. Then, C/EBPα and PIK3C2A were knockdown or overexpressed by siRNA or plasmid transfection, respectively. Rapamycin or 3-MA was utilized to stimulate and restrain autophagy of VSMCs, respectively. Western blot was used to evaluate the expression levels of C/EBPα, PIK3C2A, LC3, Beclin-1, p62, MMP-2, MMP-9, α-SMA, SM-MHC, and OPN. The pathological status of aortic ring was evaluated by stretch stress, and ChIP assay was used to analyze the binding between C/EBPα and PIK3C2A. C/EBPα shRNA was injected into tail vein to observe the effect of C/EBPα knockdown in vivo on phenotype, autophagy of aortic vascular tissue by immunohistochemical staining and Western blot. Results: The protein levels of C/EBPα, PIK3C2A, MMP-2, MMP-9, and LC3 in the aorta of AD rats were all upregulated significantly. C/EBPα and rapamycin promoted notable upregulation of the synthesized proteins (OPN), PIK3C2A, matrix metalloproteinases, LC3, and Beclin-1 in VSMCs, while suppressed contractile proteins (α-SMA and SM-MHC) and p62. The opposite results were observed in the C/EBPα-knockdown VSMCs, PIK3C2A-knockdown VSMCs, or VSMCs treated with 3-MA. C/EBPα, PIK3C2A, and LC3 were dramatically upregulated by the stimulation of 3 g and 5 g stretch stress. The downregulated contractile proteins, upregulated synthetic proteins, activated autophagy, and aggravated pathological state in 5 g stretch stress-treated aortic rings were significantly reversed by the knockdown of C/EBPα. ChIP results indicated that there was a binding site for C/EBPα in the promoter of PIK3C2A. C/EBPα also downregulated α-SMA level and upregulated OPN levels in AD rats in vivo. Conclusion: Our data indicated that during the development of AD, C/EBPα regulated the transition of VSMC phenotype and extracellular matrix remodeling by activating autophagy through regulating the transcriptional activity of PIK3C2A promoter.


Assuntos
Aneurisma Dissecante , Músculo Liso Vascular , Fosfatidilinositol 4-Fosfato 3-Quinase/metabolismo , Aneurisma Dissecante/genética , Animais , Autofagia/genética , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteína Beclina-1/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/farmacologia , Células Cultivadas , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Fenótipo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Sirolimo/farmacologia , Ativação Transcricional
16.
Int J Mol Sci ; 23(18)2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36142326

RESUMO

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.


Assuntos
Antineoplásicos , Produtos Biológicos , Glioblastoma , Glioma , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Produtos Biológicos/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioma/tratamento farmacológico , Isotiocianatos/farmacologia , Isotiocianatos/uso terapêutico , Metaloproteinase 9 da Matriz , Camundongos , Camundongos Nus , Proteína de Sequência 1 de Leucemia de Células Mieloides , Compostos Fitoquímicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteína X Associada a bcl-2
17.
Int J Biol Sci ; 18(14): 5575-5590, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36147460

RESUMO

Colorectal cancer (CRC) is an aggressive malignancy with poor prognosis. It is imperative to elucidate the potential molecular mechanisms that regulate CRC cell aggressiveness. In present study, the transient receptor potential melastatin 4 (TRPM4), a calcium-activated nonselective cation channel, is downregulated in CRC as a novel methylated tumor suppressor gene (TSG). The reduced mRNA level of TRPM4 is due to the epigenetic methylation of its promoter CpG island (CGI). Moreover, ectopic expression of TRPM4 inhibited tumor growth and metastasis both in vitro and in vivo. Our experiments also demonstrate that TRPM4 restructures the CRC cytoskeleton and activates the Ca2+-mediated calpain pathway through enhancing calcium influx. The western blot analysis shows that the expression of focal adhesion kinase (FAK), a calpain-mediated proteolytic substrate, is markedly suppressed after ectopic overexpression of TRPM4, besides, Akt (also known as protein kinase B, PKB), phosphatidylinositol 3-kinase (PI3K) as well as its central target mTOR have significantly decreased expression accompanied by elevated E-cadherin and restrained matrix metalloproteinases (MMP2/MMP9) expression. The inhibition of protease calpain effectively relieves the retard of FAK/Akt signals and reverses the migration suppression of TRPM4. Taken together, TRPM4, identified as a novel methylated TSG, employs intracellular Ca2+ signals to activate calpain-mediated cleavage of FAK and impede CRC migration and invasion through modulating the PI3K/Akt/mTOR signaling cascade, providing the first evidence that TRPM4 is likely to be a significant biomarker and potential target for CRC therapy.


Assuntos
Neoplasias Colorretais , Proteínas Proto-Oncogênicas c-akt , Caderinas/metabolismo , Cálcio/metabolismo , Calpaína/genética , Calpaína/metabolismo , Cátions , Movimento Celular/genética , Neoplasias Colorretais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPM
18.
Int J Biol Sci ; 18(14): 5522-5538, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36147479

RESUMO

Cathepsins play a role in regulation of cell function through their presence in the cell nucleus. However, the role of Cathepsin K (Ctsk) as an epigenetic regulator in osteoclasts remains unknown. Our data demonstrated that Ctsk-/-Mmp9-/- mice have a striking phenotype with a 5-fold increase in bone volume compared with WT. RNA-seq analysis of Ctsk-/- , Mmp9-/- and Ctsk-/-/Mmp9-/- osteoclasts revealed their distinct functions in gene expression regulation, including reduced Cebpa expression, increased Nfatc1 expression, and in signaling pathways activity regulation. Western blots and qPCR data validated these changes. ATAC-seq profiling of Ctsk-/- , Mmp9-/-, and Ctsk-/-/Mmp9-/- osteoclasts indicated the changes resulted from reduced chromatin openness in the promoter region of Cebpa and increased chromatin openness in Nfatc1 promoter in Ctsk-/-/Mmp9-/- osteoclasts compared to that in osteoclasts of WT, Ctsk/- and Mmp9-/- . We found co-localization of Ctsk with c-Fos and cleavage of H3K27me3 in wild-type osteoclasts. Remarkably, cleavage of H3K27me3 was blocked in osteoclasts of Ctsk-/- and Ctsk-/-/Mmp9-/- mice, suggesting that Ctsk may epigenetically regulate distinctive groups of genes' expression by regulating proteolysis of H3K27me3. Ctsk-/-/Mmp9-/- double knockout dramatically protects against ovariectomy induced bone loss. We found that Ctsk may function as an essential epigenetic regulator in modulating levels of H3K27me3 in osteoclast activation and maintaining bone homeostasis. Our study revealed complementary and unique functions of Ctsk as epigenetic regulators for maintaining osteoclast activation and bone homeostasis by orchestrating multiple signaling pathways and targeting both Ctsk and Mmp9 is a novel therapeutic approach for osteolytic diseases such as osteoporosis.


Assuntos
Reabsorção Óssea , Catepsina K , Metaloproteinase 9 da Matriz , Osteoclastos , Animais , Reabsorção Óssea/metabolismo , Catepsina K/genética , Diferenciação Celular , Cromatina/metabolismo , Feminino , Expressão Gênica , Histonas/metabolismo , Homeostase , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Knockout , Ligante RANK/metabolismo
19.
Adv Gerontol ; 35(3): 408-412, 2022.
Artigo em Russo | MEDLINE | ID: mdl-36169369

RESUMO

The development of diabetic retinopathy is associated with matrix metalloproteinases, but they are rarely used to predict this pathology. The aim of the study was to predict the development of non-proliferative diabetic retinopathy in old age by the level of matrix metalloproteinases in blood plasma. The main study group consisted of 63 patients aged 60-74 years with type 2 diabetes mellitus and non-proliferative diabetic retinopathy, the control was 56 patients of the same age with type 2 diabetes mellitus and the absence of diabetic retinopathy and other ophthalmopathology at present and in the anamnesis. Examination of patients of both groups included: tonometry, visiometry, standard fundus photoregistration, optical coherence tomography, optical coherence tomography-A, fluorescent angiography. Determination of matrix metalloproteinases was carried out by the method of solid-phase enzyme immunoassay. There was a statistically significant increase in matrix metalloproteinase-9 in the main group of patients to 55,7±2,6 ng/ml versus 40,2±1,9 ng/ml in the age control, matrix metalloproteinase-2 to 269,8±4,2 ng/ml versus 221,9±3,6 ng/ml, respectively. Based on the level of matrix metalloproteinases-2 (X1) and -9 (X2) in the blood, a regression model was created by the regression method to predict the development of diabetic retinopathy, having the form Y=28,315+3,892·X1+2,453·X2, which will allow detecting the disease at an early stage.


Assuntos
Diabetes Mellitus Tipo 2 , Retinopatia Diabética , Idoso , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/etiologia , Retinopatia Diabética/patologia , Humanos , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Metaloproteinases da Matriz , Prognóstico
20.
Sci Adv ; 8(39): eabo5525, 2022 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-36170363

RESUMO

Intracellular gap (iGap) formation in liver sinusoidal endothelial cells (LSECs) is caused by the destruction of fenestrae and appears under pathological conditions; nevertheless, their role in metastasis of cancer cells to the liver remained unexplored. We elucidated that hepatotoxin-damaged and fibrotic livers gave rise to LSECs-iGap formation, which was positively correlated with increased numbers of metastatic liver foci after intrasplenic injection of Hepa1-6 cells. Hepa1-6 cells induced interleukin-23-dependent tumor necrosis factor-α (TNF-α) secretion by LSECs and triggered LSECs-iGap formation, toward which their processes protruded to transmigrate into the liver parenchyma. TNF-α triggered depolymerization of F-actin and induced matrix metalloproteinase 9 (MMP9), intracellular adhesion molecule 1, and CXCL expression in LSECs. Blocking MMP9 activity by doxycycline or an MMP2/9 inhibitor eliminated LSECs-iGap formation and attenuated liver metastasis of Hepa1-6 cells. Overall, this study revealed that cancer cells induced LSEC-iGap formation via proinflammatory paracrine mechanisms and proposed MMP9 as a favorable target for blocking cancer cell metastasis to the liver.


Assuntos
Células Endoteliais , Neoplasias Hepáticas , Actinas/metabolismo , Animais , Doxiciclina/metabolismo , Células Endoteliais/metabolismo , Humanos , Interleucina-23/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos , Fator de Necrose Tumoral alfa/metabolismo
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