RESUMO
The development of atherosclerosis (AS) is a multifactorial process in which elevated plasma cholesterol levels play a central role. As a new class of players involved in AS, the regulation and function of microRNAs (miR) in response to AS remain poorly understood. This study analyzed the effects of miR-1 (antagomir and mimic) on endothelial permeability and myosin light chain kinase (MLCK) expression and activity in the artery wall of apoE knock-out mice after feeding them a high-cholesterol diet. Further, we tested to determine whether that effects are involved in ERK phosphorylation. Here, we show that a high-cholesterol diet induces a significant decrease of miR-1 expression. Histopathologic examination demonstrated that miR-1 antagomir enhances endothelial permeability induced by high cholesterol and miR-1 mimic attenuated endothelial barrier dysfunction. Consistent with endothelial permeability, Western blotting, qPCR, and γ-(32)P-ATP phosphate incorporation showed that MLCK expression and activity were further increased in miR-1 antagomir-treated mice and decreased in miR-1 mimic-treated mice compared with those of mice receiving control miR. Further mechanistic studies showed that high-cholesterol-induced extracellular signal regulated kinase (ERK) activation was enhanced by miR-1 antagomir and attenuated by miR-1 mimic. Collectively, those results indicate that miR-1 contributes to endothelial barrier function via mechanisms involving not only MLCK expression and activity but also ERK phosphorylation.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/prevenção & controle , Dieta Hiperlipídica/efeitos adversos , Células Endoteliais/metabolismo , MicroRNAs/genética , Animais , Aorta/metabolismo , Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Permeabilidade da Membrana Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Interferência de RNARESUMO
Compared with conventional drilling (CD), ultrasonic vibration-assisted drilling(UVAD) is experimentally proven a promising method to reduce the cutting temperature. But sometimes cutting temperature also becomes higher in UVAD than in CD. To further make clear the cutting temperature mechanisms in UVAD, this study aims to study the effect of tool's ultrasonic vibration on the cutting heat generation and heat dissipation at a relatively micro level. Firstly, drilling experiments are designed to explore the variations of cutting heat under different ultrasonic vibrations. Then, to analyze the influence of ultrasonic vibration on the cutting heat theoretically, a kinematic model is developed to describe the dynamic contact between the cutting edge and workpiece in UVAD. Besides, a cutting heat analysis model based on the contact characteristics in UVAD is proposed to study and compare the variations of cutting heat generation. The effect of ultrasonic vibration on the cutting heat generation, heat dispassion, and the resultant cutting temperature under different machining in UVAD conditions are discussed. It is indicated from the theoretical analysis that more cutting heat tends to be produced due to the significantly increased sliding velocity on the cutting edge-workpiece interface when the ultrasonic vibration is applied. The analysis agrees with the experimental results that the cutting temperature in dry UVAD is higher than in dry CD. But on the other hand, ultrasonic vibration can also improve the lubrication and cooling effect under appropriate machining conditions, which is beneficial to the reduction in cutting temperature. The investigation shows the multifaceted influences of ultrasonic vibration on the cutting temperature in the drilling process in detail, which provides a reference for UVAD parameter optimization.
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The development of hypercholesterolemia is a multifactorial process in which elevated plasma cholesterol levels play a central role. This study analyzed the variability of the expression and activity of myosin light chain kinase (MLCK) and endothelial permeability in the artery wall of rabbits after feeding the animals with a normal or a high-cholesterol diet. Hypercholesterolemia was induced by a high-cholesterol diet for 4 weeks. Aortas were removed and analyzed for endothelial permeability and MLCK expression. Samples of the arterial media were analyzed for MLCK activity and expression. A selective MLCK inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7) were used in hypercholesterolemia rabbit (1 mg/kg body weight). The aortas of high-cholesterol diet rabbits showed an increase in MLCK expression and activity (nearly threefold compare with control) as well as endothelial permeability. ML7 inhibit MLC phosphorylation and MLCK activity (nearly twofold compare with control) and endothelial permeability stimulated by cholesterol. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and activity through which endothelial permeability is increased.
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Aorta/enzimologia , Aorta/fisiopatologia , Permeabilidade Capilar/fisiologia , Hipercolesterolemia/enzimologia , Quinase de Cadeia Leve de Miosina/metabolismo , Túnica Íntima/enzimologia , Túnica Íntima/fisiopatologia , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Azepinas/farmacologia , Western Blotting , Permeabilidade Capilar/efeitos dos fármacos , Hipercolesterolemia/patologia , Hipercolesterolemia/fisiopatologia , Masculino , Naftalenos/farmacologia , Fosforilação/efeitos dos fármacos , Coelhos , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologiaRESUMO
BACKGROUND: Microribose nucleic acids (miRNAs) are implicated in the progression of lung adenocarcinoma. MicroRNA-345-5p (miR-345-5p) is a recently identified anti-oncogene in some human cancers, but its functional role and possible molecular mechanism in lung adenocarcinoma remain unknown. This study aimed to identify the biological function and underlying mechanism of miR-345-5p in lung adenocarcinoma cells. METHODS: In this study, lung adenocarcinoma tissues and adjacent tissues were collected in the First Affiliated Hospital of Anhui Medical University between April 2016 and February 2017. The expression of miR-345-5p and ras homolog family member A (RhoA) in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines (A549, H1650, PC-9, and H441) was detected by reverse transcription quantitative polymerase chain reaction analysis. Functional assays including colony formation, flow cytometry analysis, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of lung adenocarcinoma cells. In addition, RNA pulldown and luciferase reporter assays were conducted to evaluate the relationship between miR-345-5p and RhoA. Difference between the two groups was analyzed with Student's t test, while that among multiple groups was analyzed with one-way analysis of variance. RESULTS: MiR-345-5p expression displayed lower level in lung adenocarcinoma tissues (0.241 ± 0.095 vs.1.000 ± 0.233, t = 19.247, P < 0.001) and cell lines (F = 56.992, P < 0.001) than control tissues and cells. Functional experiments demonstrated that upregulation of miR-345-5p inhibited the malignant phenotypes of lung adenocarcinoma cells via suppressing cell proliferation, migration, invasion, and facilitating cell apoptosis. Additionally, RhoA was verified to be the downstream target of miR-345-5p. Expression of RhoA was downregulated by overexpression of miR-345-5p in PC-9 (0.321 ± 0.047 vs. 1.000 ± 0.127, t = 8.536, P < 0.001) and H1650 (0.398 ± 0.054 vs. 1.000 ± 0.156, t = 4.429, P = 0.011) cells. Rescue assays revealed that overexpression of RhoA rescued the suppressive effects of miR-345-5p upregulation on proliferation, migration, and invasion of lung adenocarcinoma cells. Further, miR-345-5p was found to regulate the Rho/Rho-associated protein kinase (ROCK) signaling pathway by downregulation of RhoA in lung adenocarcinoma cells. CONCLUSIONS: MiR-345-5p plays a tumor suppressor role in lung adenocarcinoma cells by downregulating RhoA to inactivate the Rho/ROCK pathway.
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Adenocarcinoma de Pulmão , Neoplasias Pulmonares , MicroRNAs , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , MicroRNAs/genética , Regulação para Cima/genética , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Melatonin, the main secretary product of the pineal gland, is potentially effective in the prevention of a number of diseases in which free radical processes are involved. The development of hypercholesterolemia is a multifactorial process in which elevated oxidized low-density lipoprotein (ox-LDL) levels play a central role. The purpose of this study was to test whether melatonin prevents ox-LDL-induced increase of myosin light chain kinase (MLCK) activation and expression in human umbilical vein endothelial cells (HUVECs) through extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signal transduction. HUVEC were cultured in vitro and treated with ox-LDL, melatonin, and PD98059 (a selective inhibitor of ERK), respectively. The expression, transcription, and activity of MLCK were measured by western blot, immunohistochemistry, reverse transcription-polymerase chain reaction and gamma-(32)P-adenosine triphosphate (ATP) incorporation, respectively. The results showed that the expression and activity of MLCK were increased in ox-LDL-treated HUVECs and this was decreased by melatonin and PD98059. The expression and activity of MLCK induced by ox-LDL was associated with the phosphorylation of ERK. These results indicate for the first time that hypercholesterolemia may be associated with MLCK expression and the activity which can be reduced by melatonin through ERK/MAPK signal transduction.
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Lipoproteínas LDL/metabolismo , Sistema de Sinalização das MAP Quinases , Melatonina/uso terapêutico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Análise de Variância , Antioxidantes , Western Blotting , Células Cultivadas , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Veias UmbilicaisRESUMO
AIM: To investigate the expression frequency of endocan in colorectal cancer and analyze the relationship between endocan expression and clinical parameters and to study the role of endocan in colorectal carcinogenesis. METHODS: Expression of endocan in 72 tumor tissue samples of colorectal cancer as well as in 27 normal mucous membrane tissue samples was analyzed using in situ hybridization, immunohistochemistry on tissue microarray, Western blot and reverse-transcript polymerase chain reaction (RT-PCR). RESULTS: The expression of endocan was higher in normal colon and rectum tissue samples than in cancerous tissue samples (mRNA = 92.6%, protein = 36%), and was lower in colorectal cancer tissue samples (mRNA = 70.4%, protein = 36.1%). No correlation was found between staining intensity and clinical parameters such as sex, age, tumor size and TNM stage. However, the expression of endocan was positively correlated with the tissue differentiation in colorectal cancer. CONCLUSION: The expression of endocan is down-regulated in colorectal cancer and is positively correlated with the tissue differentiation in colorectal cancer, suggesting that the expression of endocan is associated with development and differentiation of colorectal cancer.
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Adenocarcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteoglicanas/metabolismo , Adenocarcinoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Regulação para Baixo , Feminino , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteoglicanas/genética , RNA Mensageiro/metabolismo , Reto/metabolismo , Reto/patologia , Adulto JovemRESUMO
The present study aimed to investigate the effects of nucleotide-binding domain leucine-rich repeat protein (NLRP)1/NLRP3 inflammasome pathways on latent viral infection of the respiratory tract. A total of 55 BALB/c mice were assigned to the control, bleomycin (BLM)treated, murine cytomegalovirus (MCMV), MCMV+BLM and MCMV+BLM+CD4+ Tcell groups. The viral loads were detected in the salivary glands, kidney, liver and lung tissues via polymerase chain reaction (PCR). The weight, lung coefficient and hydroxyproline (HYP) were detected. HE and Masson staining were performed to score for alveolitis and degree of pulmonary fibrosis. Reverse transcriptionquantitative PCR and western blot were applied to assess the expression levels of the NLRP inflammasome components caspase1, interleukin (IL)1ß and IL18. ELISA was used to evaluate the expression levels of caspase1, tumor necrosis factor (TNF)α, IL1ß and IL18. The weight of the mice decreased, and the lung coefficient and HYP content increased in the BLM, MCMV, MCMV+BLM and MCMV+BLM+CD4+ Tcell groups compared with those in the control group. Compared with the control group, mice in the BLM, MCMV+BLM and MCMV+BLM+CD4+ Tcell groups had obviously increased alveolitis and degrees of pulmonary fibrosis, increased mRNA expression levels of caspase1, IL1ß and IL18, and increased protein expression levels of caspase1(p20), mature IL1ß and mature IL18. The values in the MCMV+BLM group were also higher than those in the BLM group and those in the MCMV+BLM+CD4+ Tcell group. The serum levels of caspase1, TNFα, IL1ß and IL18 in the serum of mice in the MCMV+BLM group were significantly higher than those in the BLM group. Compared with the MCMV+BLM group, the MCMV+BLM+CD4+ Tcell group had decreased levels of caspase1, TNFα, IL1ß and IL18 (all P<0.05). These results demonstrated that the activation of the NLRP1 and NLRP3 inflammasome pathways may contribute to pulmonary fibrosis caused by latent MCMV infection in mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Muromegalovirus/patogenicidade , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Bleomicina/farmacologia , Caspases/genética , Caspases/metabolismo , Hidroxiprolina/metabolismo , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To investigate the protein and mRNA expression of osteopontin (OPN) in the lung cancer tissue and explore the roles thereof in the development and progression of lung cancer. METHODS: Immunohistochemistry and in situ hybridization were used to detect the protein and mRNA expression of OPN in 57 specimens of lung cancer tissue, 30 specimens of inflammatory pseudotumors and 20 specimens of pulmonary bulla, all obtained during operation. RESULTS: The OPN protein expression rate of the lung cancer tissue was 57.9% (33/57) , significantly higher than that of the inflammatory pseudotumor (16.7%, 5/30, chi2 = 13.581, P = 0.000). The OPN mRNA expression rate in the lung cancer tissue was 71.9% (41/57), significantly higher than that of the inflammatory pseudotumor (30.0%, 9/30, chi2 = 14. 138, P = 0.000). All the 20 specimens of pulmonary bullae were negative in the expression of OPN, both at the protein and mRNA levels. The OPN protein and mRNA expression rates of the lung cancer tissues with lymph node metastasis were 71.1% (27/38) and 86.8% (33/38) respectively, both significantly higher than those of the lung cancer tissue without lymph node metastasis [31.6% (6/19) and 42.1% (8/19) respectively, chi2 = 6.558, P = 0.010, and chi2 = 10.438, P = 0.001]. The OPN protein and mRNA expression rates of the non-small lung cancer tissues were 68.1% (32/47) and 78.7% (37/47) respectively, both significantly higher than those of the small lung cancer tissues [10% (1/10) and 25% (4/10) respectively, chi2 = 11.412, P = 0.001, and chi2 = 6.124, P = 0.013]. The OPN protein expression was positively correlated with the OPN mRNA expression in the lung cancer tissues (r = 0.623, P = 0.001). The 57 patients with lung cancer after surgery were followed up for 28 (24-40) months, 8 of the 32 patients with both positive OPN protein and mRNA expression had recurrence and 13 patients had a distant metastasis, while only 1 of the 15 cases negative in both OPN protein and mRNA expression showed recurrence (chi2 = 14.258, P = 0.000). 12 patients died in the both positive expression group but no patient died in the both negative expression group (chi2 = 7.554, P = 0.006). CONCLUSION: Over-expressions of OPN protein and OPN mRNA are found in lung cancer tissues and their expressions are correlated to the prognosis and metastasis of lung cancer.
Assuntos
Neoplasias Pulmonares/patologia , Osteopontina/genética , Adulto , Idoso , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Osteopontina/metabolismo , Granuloma de Células Plasmáticas Pulmonar/genética , Granuloma de Células Plasmáticas Pulmonar/metabolismo , Granuloma de Células Plasmáticas Pulmonar/patologia , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. MATERIALS AND METHODS: The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). RESULTS: The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. CONCLUSIONS: Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.
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Antioxidantes/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melatonina/farmacologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Regulação para Baixo , Humanos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.
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Carcinoma Hepatocelular/genética , Genes p53 , Neoplasias Hepáticas/genética , Sequência de Bases , China , DNA de Neoplasias/genética , Éxons , Humanos , Dados de Sequência Molecular , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , Homologia de Sequência do Ácido NucleicoRESUMO
AIM: To analyze loss of heterozygosity (LOH) and homozygous deletion on p53 gene (exon2-3, 4 and 11), chromosome 10q22-10q23 and 22q11.2 -22q12.1 in human hepatocellular carcinoma (HCC). METHODS: PCR and PCR-based microsatellite polymorphism analysis techniques were used. RESULTS: LOH was observed at D10S579 (10q22-10q23) in 4 of 20 tumors (20%), at D22S421 (22q11.2-22q12.1) in 3 of 20(15%), at TP53.A (p53 gene exon 2-3) in 4 of 20(20%), at TP53.B (p53 gene exon 4) in 6 of 20(30%), and at TP53.G (p53 gene exon 11) in 0 of 20(0%). Homozygous deletion was detected at 10q22-10q23(8/20; 40%), 22q11.2-22q12.1(8/20; 40%), p53 gene exon 2-3(0/20;0%), p53 gene exon 4(6/20; 30%), and p53 gene exon 11(2/20; 10%). CONCLUSION: There might be unidentified tumor suppressor genes on chromosome 10q22-10q23 and 22q11.2-22q12.1 that contribute to the pathogenesis and development of HCC.
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Carcinoma Hepatocelular/genética , Cromossomos Humanos Par 10 , Cromossomos Humanos Par 22 , Neoplasias Hepáticas/genética , Perda de Heterozigosidade , Proteína Supressora de Tumor p53/genética , Cromossomos Humanos Par 17 , Éxons , Deleção de Genes , Homozigoto , Humanos , Repetições de MicrossatélitesRESUMO
AIM: To study the distribution and expression of non-muscle myosin light chain kinase (nmMLCK) in rabbit livers. METHODS: Human nmMLCK N-terminal cDNA was amplified by polymerase chain reaction (PCR) and was inserted into pBKcmv to construct expression vectors. The recombinant plasmid was transformed into XL1-blue. Expression protein was induced by IPTG and then purified by SDS-PAGE and electroelution, which was used to prepare the polycolonal antibody to detect the distribution and expression of nmMLCK in rabbit livers with immunofluorescene techniques. RESULTS: The polyclonal antibody was prepared, by which nmMLCK expression was detected and distributed mainly in peripheral hepatocytes. CONCLUSION: nmMLCK can express in hepatocytes peripherally, and may play certain roles in the regulation of hepatic functions.
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Fígado/enzimologia , Quinase de Cadeia Leve de Miosina/genética , Animais , Sequência de Bases , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Fígado/citologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/análise , Reação em Cadeia da Polimerase , CoelhosRESUMO
The myosin light chain kinase (MLCK) is important in regulating the smooth muscle contraction. The pBKrsv-MLCK was obtained by inserting MLCK cDNA into pBKrsv polylinker and transfected into NIH 3T3 cells. The results revealed that the transfected cells can express chicken MLCK identified by DNA-PCR, RT-PCR and Western blot. The expressed MLCK has high activity, thus providing the basis for further investigation of the signal transduction and regulation of smooth muscle contraction of MLCK.
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OBJECTIVE: To study the effect of vitamin E (Vit E) on the myosin light chain kinase(MLCK) activity and the endothelial permeability of the artery in atherosclerotic rabbits. METHODS: The MLCK activity of rabbit artery was measured by incorporation of gamma-(32)P. The endothelial permeability was accessed by immunofluorescence. RESULTS: The model of atherosclerosis was established after rabbits were fed with cholesterol for four weeks. The activity of MLCK increased markedly, and there was significantly statistical difference compared with the normal control (P<0.05). When the rabbits were fed with cholesterol for twelve weeks or with cholesterol and Vit E for twelve weeks, the activity of MLCK did not change markedly, and there was no statistical difference compared with the normal control, respectively (P>0.05). The permeability of arterial wall was increased after the rabbits were fed with cholesterol for four weeks, and the permeability increased even more obviously after the rabbits were fed with cholesterol for twelve weeks. The permeability appeared to be decreased when Vit E was added into the cholesterol feeding. CONCLUSION: The change in integrity of arterial wall may be associated with the increase of the activity of MLCK. Vit E may decrease the MLCK activity. Vit E may decrease the endothelial permeability of atherosclerotic rabbits.
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Quinase de Cadeia Leve de Miosina/efeitos dos fármacos , Vitamina E/farmacologia , Vitaminas/farmacologia , Animais , Artérias , Endotélio Vascular/efeitos dos fármacos , Humanos , Permeabilidade , CoelhosRESUMO
AIMS: To explore the effect and probable mechanism of a synthetic retinoid 4-amino-2-tri-fluoromethyl- phenyl ester (ATPR) on apoptosis of MDA-MB-231 breast cancer cells. MATERIALS AND METHODS: MTT assays were performed to measure the proliferation of MDA-MB-231 cells treated with different concentrations of all- trans retinoic acid (ATRA) and ATPR. Morphologic changes were observed by microscopy. The apoptosis rates and cell cycling of MDA-MB-231 cells treated with ATRA or ATPR were assessed using flow cytometry analysis. Expression of retinoic acid receptor and phosphorylation of ERK, JNK, p38 proteins were detected by Western blotting. RESULTS: Treatment of the cells with the addition of 15 µmol/L ATPR for 48 h clearly demonstrated reduced cell numbers and deformed cells, whereas no changes in the number and morphology were observed after treatment with ATRA. The apoptosis rate was 33.2% after breast cancer MDA-MB-231 cells were treated by ATPR (15 µmol/L) whereas ATRA (15 µmol/L) had no apoptotic effect. ATPR inhibited the phosphorylation of ERK, JNK, and p38 while ATRA had no significant effect. ATPR inhibited the expression of BiP and increased the expression of Chop at the protein level compared with control groups, ATRA and ATPR both decreased the protein expression of RXR α, ATPR reduced the protein expression of RARß and RXRß while ATRA did not decrease RARß or RXRß. CONCLUSIONS: ATPR could induce apoptosis of breast cancer MDA-MB-231 cells, possible mechanisms being binding to RARß/RXRß heterodimers, then activation of ER stress involving the MAPK pathway.
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Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Transdução de Sinais/efeitos dos fármacos , Tretinoína/química , Tretinoína/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Fosforilação/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
AIM: To observe the effects of a novel all-trans retinoid acid (ATRA) derivative, N-(3-trifluoromethyl-phenyl)- retinamide (ATPR), on lung adenocarcinoma A549 cells and to explore the potential mechanism of ATPR inhibiting of A549 cell migration. MATERIALS AND METHODS: The cytotoxicity of ATRA and ATPR on A549 cells was assessed using MTT assay. Wound healing assays were used to analyze the influences of ATRA, ATPR, ML-7 (a highly selective inhibitor of myosin light chain kinase (MLCK)), PMA (an activator of MAPKs) and PD98059 (a selective inhibitor of ERK1/2) on the migration of A549 cells. Expression of MLCK and phosphorylation of myosin light chain (MLC) were assessed by Western blotting. RESULTS: ATRA and ATPR inhibited the proliferation of A549 cells in a dose- and time-dependent manner, and the effect of ATPR was much more remarkable compared with ATRA. Relative migration rate and migration distance of A549 cells both decreased significantly after treatment with ATPR or ML-7. The effect on cell migration of PD98059 combining ATPR treatment was more notable than that of ATPR alone. Moreover, compared with control groups, the expression levels of MLCK and phosphorylated MLC in A549 cells were both clearly reduced in ATRA and ATPR groups. CONCLUSIONS: ATPR could suppress the migration and invasion of A549 cells, and the mechanism might be concerned with down- regulating the expression of MLCK in the ERK-MAPK signaling pathway, pointing to therapeutic prospects in lung cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Tretinoína/análogos & derivados , Tretinoína/farmacologia , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
OBJECTIVE: The present study was designed to investigate the probable mechanisms of synthetic retinoid 4-amino-2-tri-fluoromethyl-phenyl ester (ATPR) inhibition of the proliferation and migration of A549 human lung carcinoma cells. MATERIALS AND METHODS: After the A549 cells were treated with different concentrations of ATPR or all-trans retinoic acid (ATRA) for 72 h, scratch-wound assays were performed to assess migration. Immunofluorescence was used to determine the distribution of CAV1 and RXRα, while expression of CAV1, MLCK, MLC, P38, and phosphorylation of MLC and P38 were detected by Western blotting. RESULTS: ATPR could block the migration of A549 cells. The relative migration rate of ML-7 group had significantly decreased compared with control group. In addition, ATPR decreased the expression of a migration related proteins, MLCK, and phosphorylation of MLC and P38. ATPR could also influence the expression of RARs or RXRs. At the same time, CAV1 accumulated at cell membranes, and RXRα relocated to the nucleus after ATPR treatment. CONCLUSIONS: Caveolae may be implicate in the transport of ATPR to the nucleus. Change in the expression and distribution of RXRα may be implicated in ATPR inhibition of A549 cell proliferation. The mechanisms of ATPR reduction in A549 cell migration may be associated with expression of MLCK and phosphorylation of MLC and P38.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Retinoides/farmacologia , Cicatrização/efeitos dos fármacos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Western Blotting , Caveolina 1/metabolismo , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Naftalenos/farmacologia , Receptores X de Retinoides/metabolismo , Transdução de Sinais , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
The aim of the present study was to synthesize a series of retinamide derivatives using all-trans retinoic acid (ATRA) as raw material and observe their effects on the differentiation and apoptosis of human lung adenocarcinoma A549 cells. Four new synthesized ATRA retinamide derivatives were structurally confirmed by spectral analysis, including (1)H-NMR, (13)C-NMR, and MS. The results showed that the new ATRA retinamide derivatives significantly decreased the carcinoembryonic antigen secretion of A549 cells, significantly decreased the proliferation of A549 cells in a dose- and time-dependent manner, and promoted the apoptosis of A549 cells compared with ATRA. The Western blot assay indicated that the expression of Bcl-2 was decreased more in A549 cells treated with N-(3-trifluoromethylphenyl) retinamide than that in A549 cells treated with ATRA. The results also showed that the effects of N-(3-trifluoromethyl-phenyl) retinamide on differentiation and apoptosis were the strongest among the newly synthesized ATRA retinamide derivatives. Our results suggested that the effects of novel ATRA retinamide derivatives on increasing the differentiation, decreasing the proliferation, and promoting the apoptosis of A549 cells were greater than those of ATRA. The apoptosis of A549 cells induced by N-(3-trifluoromethylphenyl) retinamide may be related to downregulating the expression of Bcl-2.