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1.
Chinese Acupuncture & Moxibustion ; (12): 765-770, 2020.
Article in Chinese | WPRIM | ID: wpr-826658

ABSTRACT

OBJECTIVE@#To observe the effect of electroacupuncture (EA) preconditioning on the expressions of tyrosine kinase Lyn and spleen tyrosine kinase (Syk) in mast cells of subcutaneous loose connective tissue in the rats with urticaria and explore the potential biological mechanism of EA in the intervention of urticaria.@*METHODS@#A total of 32 SD rats were randomized into a blank group, a model group, an EA group and a positive medication group, 8 rats in each one. Except of the blank group, the passive cutaneous anaphylaxis (PCA) was adopted to prepare the model of urticaria in the rats of the rest three groups. In the EA group, EA was applied to bilateral "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36), with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, once daily, for 20 min each time, consecutively for 7 days. In the positive medication group, loratadine (1 mg•kg•d) was for intragastric administration, once daily, consecutively for 7 days. The samples were collected for index detection 30 min after PCA antigen challenge in the rats of each group. Spectrophotometer was adopted to determine the effusion quantity of Evans blue in the allergized site of skin. HE staining was used to observe the morphological changes in the allergized site of skin. Toluidine blue staining was provided to observe mast cell degranulation in subcutaneous loose connective tissue in the allergized site of skin. Immunohistochemistry was applied to determine the protein expressions of Lyn and Syk during degranulation of mast cells.@*RESULTS@#In the rats of the odel group, the eipdermis of allergized site was thickening, cells were disorganized in hierarchy and inflammatory cells were infiltrated largely in the dermis. In the positive medication group and the EA group, the epidermis was getting thin, cell arrangement was clear and the inflammatory cell infiltration was obviously alleviated as compared with the model group. Compared with the blank group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all increased in the model group (<0.01). Compared with the model group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all reduced in the EA group and the positive medication group (<0.01). Compared with the positive medication group, the degranulation rate of mast cells was increased significantly in the EA group (<0.01).@*CONCLUSION@#Electroacupuncture at "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36) reduces vascular permeability and gives play to the role of anti-allergy by the way of regulating and controlling the degranulation of mast cells in the rats with urticaria and the effect mechanism of electroacupuncture may be related to the inhibition of protein expressions of Lyn and Syk in mast cells.


Subject(s)
Animals , Rats , Acupuncture Points , Connective Tissue , Metabolism , Electroacupuncture , Mast Cells , Metabolism , Random Allocation , Rats, Sprague-Dawley , Syk Kinase , Metabolism , Urticaria , Therapeutics , src-Family Kinases , Metabolism
2.
Chinese Journal of Cardiology ; (12): 323-328, 2020.
Article in Chinese | WPRIM | ID: wpr-941112

ABSTRACT

Objective: To investigate whether inflammatory factor tumor necrosis factor-α (TNF-α) is involved in the electrical remodeling of cardiomyocytes by regulating ultra-rapid delayed rectifier K(+) current (I(kur)) and the role of Src kinase. Methods: H9c2 cells, embryonic cardiomyocytes of rat, were cultured in Dulbecco's modified Eagle's medium (DMEM) and atrium-derived HL-1 cells were cultured in Claycomb medium. Both H9c2 and HL-1 cells were cultured at 37 ℃ with 5% CO(2). Cells cultured in normal conditions without additional treatment served as control group. Experimental groups were treated with different concentration of TNF-α (25 or 50 or 100 ng/ml) for 24 hours. To study whether Src specific inhibitor PP1 could abrogate the effect of TNF-α, cells were pre-treated with 10 μmol/L PP1 for 1 hour, followed by TNF-α (100 ng/ml) for 24 hours. Western blot and the whole cell patch clamp technique were used to detect the protein expression of Kv1.5 and Src and I(kur) in each group. Results: (1) In H9c2 cells, high concentration of TNF-α treatment (100 ng/ml) significantly reduced the Kv1.5 protein expression compared with control group and TNF-α 25 ng/ml group (both P<0.05). Compared with control group, the expression of p-Src protein was higher in 25 ng/ml, 50 ng/ml, 100 ng/ml TNF-α group (all P<0.05), but there was no statistical difference in the expression of Src protein among groups (P>0.05). In addition, the current density of I(kur) was decreased in 50 ng/ml, 100 ng/ml TNF-α group (both P<0.05). Furthermore, the expression of Kv1.5 protein and the current density of I(kur) were increased in PP1+TNF-α group compared with TNF-α 100 ng/ml group (both P<0.05). There was no statistical difference in the expression of Kv1.5 protein and the current density of I(kur) between the control group and PP1+TNF-α group (both P>0.05). (2) In atrium-derived HL-1 cells, the expression of Kv1.5 protein was reduced in 100 ng/ml TNF-α group compared with control group and TNF-α 25 ng/ml group (both P<0.01). In addition, the expression of p-Src protein was increased in TNF-α 100 ng/ml group compared with control group (P<0.05), but there was no statistical difference in the protein expression of Src among groups (P>0.05). The expression of Kv1.5 protein was increased in PP1+TNF-α group compared with TNF-α 100 ng/ml group (P<0.05). Conclusion: TNF-α is involved in the pathogenesis of atrial fibrillation, probably via decreasing I(kur) current density in atrium-derived myocytes through the activation of Src kinase.


Subject(s)
Animals , Rats , Down-Regulation , Heart Atria , Myocytes, Cardiac , Tumor Necrosis Factor-alpha , src-Family Kinases
3.
Biomédica (Bogotá) ; 39(3): 547-560, jul.-set. 2019. graf
Article in Spanish | LILACS | ID: biblio-1038814

ABSTRACT

Resumen Introducción. La leptina es una hormona secretada por los adipocitos que se ha relacionado con el proceso de la transición de epitelio a mesénquima (Epithelial- Mesenchymal Transition, EMT). Promueve la migración e invasión de las células del epitelio mamario mediante la activación de las cinasas FAK y Src, un complejo regulador de vías de señalización que favorecen la expresión de las proteínas relacionadas con la formación de estructuras proteolíticas implicadas en la invasión y progresión del cáncer. Recientemente, se ha descrito que la sobreexpresión y activación de la proteína Hic-5 durante el mencionado proceso de transición, favorece la formación de los puntos de actina (indicativa de la formación y funcionalidad de los invadopodios), lo cual promueve la degradación local de los componentes de la matriz extracelular y la metástasis del cáncer. Objetivos. Evaluar el papel de las cinasas FAK y Src sobre la expresión y localización subcelular de Hic-5 y la formación de puntos de actina inducida por la leptina en la línea celular MCF10A de epitelio mamario no tumoral. Materiales y métodos. Se utilizaron los inhibidores específicos de la FAK (PF-573228) y la Src (PP2) para evaluar el papel de ambas cinasas en los niveles de expresión y localización subcelular de la proteína Hic-5 mediante Western blot e inmunofluorescencia, así como la formación de puntos de actina mediante la tinción con faloidina-TRITC en células MCF10A estimuladas con leptina. Resultados. La leptina indujo el incremento en la expresión de Hic-5 y la formación de puntos de actina. El tratamiento previo con los inhibidores de las cinasas FAK (PF-573228) y Src (PP2), promovió la disminución en la expresión de Hic-5 y de los puntos de actina en la línea celular MCF10A de epitelio mamario no tumoral. Conclusión. La leptina indujo la expresión y la localización perinuclear de Hic-5 y la formación de puntos de actina mediante un mecanismo dependiente de la actividad de las cinasas FAK y Src en las células MCF10A.


Abstract Introduction: Leptin is a hormone secreted by adipocytes that has been associated with the epithelial-mesenchymal transition (EMT). Additionally, leptin promotes the migration and invasion of mammary epithelial cells through the activation of FAK and Src kinases, which are part of a regulatory complex of signaling pathways that promotes the expression of proteins related to the formation of proteolytic structures involved in the invasion and progression of cancer. Recently, overexpression and activation of Hic-5 during the EMT have been shown to induce the formation of actin puncta; these structures are indicative of the formation and functionality of invadopodia, which promote the local degradation of extracellular matrix components and cancer metastasis. Objective: To evaluate the role of FAK and Src kinases in the expression of Hic-5 during the epithelial-mesenchymal transition induced by leptin in MCF10A cells. Materials and methods: We used specific inhibitors of FAK (PF-573228) and Src (PP2) to evaluate Hic-5 expression and subcellular localization by Western blot and immunofluorescence assays and to investigate the formation of actin puncta by epifluorescence in MCF10A cells stimulated with leptin. Results: Leptin induced an increase in Hic-5 expression and the formation of actin puncta. Pretreatment with inhibitors of FAK (PF-573228) and Src (PP2) promoted a decrease in Hic-5 expression and actin puncta formation in the non-tumorigenic mammary epithelial cell line MCF10A. Conclusion: In MCF10A cells, leptin-induced Hic-5 expression and perinuclear localization, as well as the formation of actin puncta through a mechanism dependent on the kinase activity of FAK and Src.


Subject(s)
Humans , src-Family Kinases/physiology , Leptin/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Fanconi Anemia Complementation Group C Protein/physiology , Epithelial-Mesenchymal Transition/drug effects , LIM Domain Proteins/metabolism , Pyrimidines/pharmacology , Sulfones/pharmacology , Signal Transduction , Cell Line , Actins , Quinolones/pharmacology , src-Family Kinases/antagonists & inhibitors , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Fanconi Anemia Complementation Group C Protein/antagonists & inhibitors , Epithelial-Mesenchymal Transition/physiology , Neoplasm Invasiveness
4.
Journal of Southern Medical University ; (12): 797-803, 2019.
Article in Chinese | WPRIM | ID: wpr-773530

ABSTRACT

OBJECTIVE@#To investigate the effect of SRC kinase inhibitor PP2 on the invasion and metastasis of lung cancer A549 cells and explore its molecular mechanism.@*METHODS@#MTT assay was used to evaluate the inhibitory effect of PP2 on the proliferation of A549 cells. Cell scratch and Transwell assays were performed to assess the invasion and metastatic capacity of A549 cells after treatment with 1, 2, 4, 8, and 16 μmol/L PP2 for 24 h. Western blotting was used to detect the expressions of connexin43 (Cx43) and MMP-2 in the cells after small interfering RNA (siRNA)-mediated silencing or overexpression of Cx43; the changes in the cell invasion and metastasis in response to PP2 treatment after Cx43 silencing or overexpression were investigated.@*RESULTS@#MTT assay showed that treatment with PP2 at 2, 4, 8, 16, and 32 μmol/L significantly inhibited the proliferation of A549 cells in a concentration-dependent manner. Treatments with PP2 at 1, 2, 4, 8, and 16 μmol/L for 24 h also concentration-dependently lowered the invasion and metastatic abilities of the cells ( < 0.05). At 4 and 8 μmol/L, PP2 significantly increased the expression level of Cx43 protein and decreased the expression level of MMP-2 protein. Overexpression of Cx43 significantly enhanced the inhibitory effect of PP2 on the cell invasion and metastasis, and Cx43 silencing significantly attenuated the inhibitory effect of PP2 ( < 0.05).@*CONCLUSIONS@#PP2 treatment can suppress the invasion and metastasis of A549 cells possibly by modulating the expression of Cx43.


Subject(s)
Humans , A549 Cells , Cell Line, Tumor , Cell Movement , Cell Proliferation , Connexin 43 , Lung Neoplasms , Neoplasm Invasiveness , Protein Kinase Inhibitors , src-Family Kinases
5.
The Korean Journal of Physiology and Pharmacology ; : 259-265, 2017.
Article in English | WPRIM | ID: wpr-728572

ABSTRACT

Excessive influx and the subsequent rapid cytosolic elevation of Ca²⁺ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic Ca²⁺ level in normal as well as pathological conditions. Delayed rectifier K⁺ channels (I(DR) channels) play a role to suppress membrane excitability by inducing K⁺ outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under Ca²⁺-mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of IDR channels to hyperexcitable conditions induced by high Ca²⁺ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high Ca²⁺-treatment significantly increased the amplitude of IDR without changes of gating kinetics. Nimodipine but not APV blocked Ca²⁺-induced IDR enhancement, confirming that the change of I(DR) might be targeted by Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated I(DR) enhancement was not affected by either Ca²⁺-induced Ca²⁺ release (CICR) or small conductance Ca²⁺-activated K⁺ channels (SK channels). Furthermore, PP2 but not H89 completely abolished I(DR) enhancement under high Ca²⁺ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for Ca²⁺-mediated I(DR) enhancement. Thus, SFKs may be sensitive to excessive Ca²⁺ influx through VDCCs and enhance I(DR) to activate a neuroprotective mechanism against Ca²⁺-mediated hyperexcitability in neurons.


Subject(s)
Animals , Humans , Rats , Calcium Channels , Cytosol , Kinetics , Membranes , Neurons , Nimodipine , Protein-Tyrosine Kinases , Receptors, N-Methyl-D-Aspartate , src-Family Kinases , Tyrosine
6.
Cancer Research and Treatment ; : 643-655, 2017.
Article in English | WPRIM | ID: wpr-167303

ABSTRACT

PURPOSE: KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo. MATERIALS AND METHODS: The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. RESULTS: KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. CONCLUSION: KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.


Subject(s)
Animals , Mice , Aneuploidy , Breast Neoplasms , Cell Cycle , Cell Cycle Checkpoints , Cell Line , Fluorescent Antibody Technique , Heterografts , In Vitro Techniques , Microtubules , Mitosis , src-Family Kinases , Triple Negative Breast Neoplasms , Tubulin , Wound Healing
7.
Chinese Journal of Applied Physiology ; (6): 26-31, 2016.
Article in Chinese | WPRIM | ID: wpr-254964

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of hypoxia on the human pulmonary artery smooth muscle cells two pore domain potassium channels TASK-1 and the regulation of non-receptor tyrosine kinase c-Src in this process.</p><p><b>METHODS</b>The cultured human pulmonary artery smooth muscle cells (hPASMCs) were divided into: normal group, hypoxia 30 minute group, hypoxia 6 hours group and hypoxia 48 hour group, and hypoxia 48 hour + PP2 group, hypoxia 48 hour + PP3 group, hypoxia 48 hour + bpV group. Flow cytometry was used to analyze the cell cycle, RT-PCR and Western blot technique were carried out to detect the expression changes of TASK-1 mRNA and protein in different groups.</p><p><b>RESULTS</b>(1) Cell Cycle Show: Compared with normal control group, with prolonged hypoxia, the percentages of hPASMCs in S phases of cell cycle were increased. While compared with hypoxia 48 hour group, the percentages of hypoxia 48 hour + PP2 group hPASMCs in S phases of cell cycle were decreased. The expression of TASK-1 mRNA on hPASMCs in acute hypoxia 6 hour group was increased, while the expression of TASK-1 protein on hPASMCs in the acute and chronic hypoxia group was decreased, and the expression of TASK-1 mRNA on hPASMCs in the chronic hypoxia group was decreased; After pre-incubation of a potent and selective inhibitor of the Src family of protein tyrosine kinases PP2, the expression of TASK-1 mRNA and protein in hypoxia 48 hour group was increased, however after pre-incubation of the inhibitor of the Src family of protein tyrosine phosphatase bpV, the expression of TASK-1 protein in hypoxia 48 hour group was decreased.</p><p><b>CONCLUSION</b>Hypoxia promotes human pulmonary artery smooth muscle cell proliferation, and non-receptor tyrosine kinase c-Src may participate in the expression of two pore domain potassium channels TASK-1 regulated by hypoxia. Therefore, we hypothesized that TASK-1 channels and c-Src participatein the acute and chronic hypoxic human pulmonary vasoconstriction.</p>


Subject(s)
Humans , Cell Hypoxia , Cell Proliferation , Cells, Cultured , Myocytes, Smooth Muscle , Cell Biology , Nerve Tissue Proteins , Metabolism , Potassium Channels, Tandem Pore Domain , Metabolism , Pulmonary Artery , Cell Biology , RNA, Messenger , Vasoconstriction , src-Family Kinases , Metabolism
8.
Journal of Southern Medical University ; (12): 1-7, 2016.
Article in English | WPRIM | ID: wpr-232520

ABSTRACT

<p><b>OBJECTIVE</b>Both of gp91(phox) (an isoform of nicotinamide adenine dinucleotide phosphate-reduced oxidases) and Src (a non-receptor protein tyrosine kinase) play a prominent role in mediating hypoxia/reoxygenation injury of neurons. The present study was designed to investigate the neuroprotective effect of equol, a predominant active metabolite of daidzein, against hypoxia/reoxygenation injury in rat pheochromocytoma cell line (PC12) and explore the underlying mechanisms.</p><p><b>METHODS</b>PC12 cells exposed to hypoxia/reoxygenation injury were examined for reactive oxygen species (ROS) using dihydroethidium and 2', 7'-dichlorofluorescein diacetate and analyzed for changes in lactate dehydrogenase (LDH) activity and malondialdehyde (MDA) content. The expression levels of gp91(phox) and phosphorylated Src-Tyr416 (p-Src) were measured using Western blotting.</p><p><b>RESULTS</b>Equol dose-dependently restored the cell viability and decreased LDH activity and MDA content in culture medium of PC12 cells exposed to hypoxia/reoxygenation. Pretreatment of the cells with 10(-5) and 10(-6) mol/L equol inhibited hypoxia/reoxygenation-induced increase of ROS. PC12 cells treated with equol prior to hypoxia/reoxygenation injury showed significant enhancement of the protein levels of gp91(phox) and p-Src.</p><p><b>CONCLUSION</b>Equol confers neuroprotection against hypoxia/reoxygenation injury in PC12 cells by inhibiting the generation of ROS very likely as a result of down-regulation of gp91(phox) and inhibition of Src phosphorylation.</p>


Subject(s)
Animals , Rats , Cell Hypoxia , Cell Survival , Down-Regulation , Equol , Pharmacology , L-Lactate Dehydrogenase , Metabolism , Malondialdehyde , Metabolism , Membrane Glycoproteins , Metabolism , NADPH Oxidase 2 , NADPH Oxidases , Metabolism , Neurons , Metabolism , Neuroprotective Agents , Pharmacology , PC12 Cells , Phosphorylation , Reactive Oxygen Species , Metabolism , src-Family Kinases , Metabolism
9.
China Journal of Chinese Materia Medica ; (24): 3267-3272, 2015.
Article in Chinese | WPRIM | ID: wpr-304820

ABSTRACT

To study the effect of sodium aescinate in inducing human breast cancer MCF-7 cells apoptosis and its possible mechanism. MTT assay was used to detect the inhibitory effect of sodium aescinate on the proliferation of MCF-7 cells. The morphological changes were observed under inverted microscope. DAPI nuclear staining was used to detect the changes in cell nucleus. Annexin V-FITC/PI flow cytometry was adopted to test the apoptosis rate. Changes in apoptosis-related proteins (PARP, cleaved caspase-8 and pro-caspase-3), cell survival-associated signal molecules (AKT and ERK) and their common upstream kinase SRC was detected by Western blotting. The result showed that after different concentrations of sodium aescinate were used to treat breast cancer MCF-7 cells, they inhibited the proliferation of MCF-7 cells in a dose-dependent manner, induced cell apoptosis (typical morphological changes in nucleus, significant increase in cell apoptosis rate). The expressions of cleaved PARP and caspase-8 increased, while the expression of pro-caspase-3 decreased, which further verified sodium aescinate's effect in inducing cell apoptosis. Sodium aescinate significantly inhibited the phosphorylation of cell survival-related signal molecules (AKT, ERK) and down-regulate the activation of their common up-stream kinase SRC. The findings indicated that sodium aescinate can block signals transiting to downstream molecules AKT, ERK, inhibit the proliferation of breast cancer cell MCF-7 cell apoptosis and induced cell apoptosis by suppressing the activation of SRC.


Subject(s)
Female , Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Breast Neoplasms , Drug Therapy , Genetics , Down-Regulation , Drugs, Chinese Herbal , Pharmacology , Extracellular Signal-Regulated MAP Kinases , Genetics , Metabolism , MCF-7 Cells , Proto-Oncogene Proteins c-akt , Genetics , Metabolism , Saponins , Pharmacology , Signal Transduction , Triterpenes , Pharmacology , src-Family Kinases , Genetics , Metabolism
10.
Journal of Southern Medical University ; (12): 239-243, 2015.
Article in Chinese | WPRIM | ID: wpr-239205

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of Src kinase inhibitor PP2 on hypoxia/reoxygenation (H/R) injury in rat astrocytes in vitro.</p><p><b>METHODS</b>In vitro cultured rat astrocytes were exposed to hypoxia for 8 h followed by reoxygenation for 24 h with or without pretreatment with PP2 (10 µmol/L) for 24 h before H/R injury. MTT assay and flow cytometry were used to detect the viability and apoptosis of the exposed astrocytes, respectively, and the protein expressions of Src, Bax, and Bcl-2 in the cells were determined using Western blotting.</p><p><b>RESULTS</b>PP2 pretreatment significantly increased the viability and decreased the apoptosis rate of rat astrocytes exposed to H/R injury (P<0.01). Western blotting showed that H/R injury caused increased expression of Src kinase, which was lowered by PP2 pretreatment. The ratio of Bax/bcl-2 in the astrocytes increased after H/R injury, and was significantly decreased by PP2 pretreatment (P<0.01).</p><p><b>CONCLUSION</b>PP2 protects rat astrocytes from H/R injury possibly by inhibiting the expression of Src kinase and activating the anti-apoptotic mechanisms in the cells.</p>


Subject(s)
Animals , Rats , Apoptosis , Astrocytes , Pathology , Cell Hypoxia , Cells, Cultured , Flow Cytometry , Pyrimidines , Pharmacology , src-Family Kinases
11.
Chinese Journal of Hepatology ; (12): 363-367, 2015.
Article in Chinese | WPRIM | ID: wpr-290450

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the role of Src family kinases (Fgr, Hck, Lyn) and the major protein kinase C substrate SSeCKS in non-alcoholic steatohepatitis (NASH) and determine the possible mechanism regulating differential expression.</p><p><b>METHODS</b>Kupffer cells were stimulated with CCL4 and effect on SSeCKS, Hck, Fgr, and Lyn expression was detected by real-time reverse transcription-PCR. Male Sprague-Dawley rats were used to create a NASH model by feeing a high fat diet. The modeled rats were divided into a model group and a normal group. After sacrifice, the extent of hepatic steatosis and inflammation was assessed, and the expression levels of SSeCKS and Hck, Fgr, Lyn were detected by immunohistochemical staining.</p><p><b>RESULTS</b>Expression of Lyn and Hck was decreased in the CCL4-stimulated Kupffer cells and the change in expression level was positively associated with levels of inflammatory stimuli (P < 0.01). The change in expression of SSeCKS in the CCL4-stimulated Kupffer cells was negatively correlated with inflammatory stimuli (P < 0.01). Fgr expression was very low in the unstimulated Kupffer cells and was not affected by the exposure to inflammatory stimuli. The number of inflammatory cells in the liver tissues of rars were negatively correlated with expression of Lyn, Hck and SSeCKS (P < 0.01), with low negative correlation for Lyn (r =-0.398, P < 0.01) and moderate negative correlation for Hck (r=-0.508, P < 0.01); the Lyn and Hck expression levels were highly positively correlated (r =0.942, P < 0.01).</p><p><b>CONCLUSION</b>Src family kinases (Lyn, Hck and Fgr) and SSeCKS are involved in development and progression of NASH, and their differential expression patterns are associated to a certain extent. The factors may represent potential targets of therapy for NASH-related inflammation.</p>


Subject(s)
Animals , Male , Rats , A Kinase Anchor Proteins , Cell Cycle Proteins , Fatty Liver, Alcoholic , Inflammation , Rats, Sprague-Dawley , src-Family Kinases
12.
Journal of Clinical Otorhinolaryngology Head and Neck Surgery ; (24): 789-792, 2014.
Article in Chinese | WPRIM | ID: wpr-748138

ABSTRACT

OBJECTIVE@#To investigated the effects of Src family kinases on the expression of mRNA and protein of Nav1.1 in spiral ganglion neurons.@*METHOD@#RT-PCR and Western blot techniques respectively explored the level of expression of mRNA and protein of Nav1.1 in spiral ganglion neurons by Src family kinases inhibitor.@*RESULT@#An application of the inhibitor of Src family kinases which was PP2 (10 micromol/L) and SU6656 (2 micromol/L) gived rise to the mRNA decreasing to 26% +/- 0.8% and 36% +/- 1.5% respectively (P < 0.05), and protein reducing to 39% +/- 12.5% and 53% +/- 1.7% severally (P < 0.05).@*CONCLUSION@#Administration of the inhibitor of Src family kinases could decrease the expression of mRNA and protein of Nav1.1 in spiral ganglion neurons.


Subject(s)
Animals , Male , Rats , Indoles , Pharmacology , Metabolism , Neurons , Metabolism , Pyrimidines , Pharmacology , Rats, Sprague-Dawley , Spiral Ganglion , Cell Biology , Sulfonamides , Pharmacology , src-Family Kinases , Metabolism
13.
International Journal of Oral Biology ; : 131-136, 2014.
Article in English | WPRIM | ID: wpr-102299

ABSTRACT

Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.


Subject(s)
Humans , Actins , Caveolae , Caveolin 1 , Clathrin , Endothelial Cells , GTP Phosphohydrolases , Human Umbilical Vein Endothelial Cells , Microtubules , Phosphatidylinositol 3-Kinase , Phosphotransferases , Polymerization , Polymers , Porphyromonas gingivalis , RNA, Small Interfering , src-Family Kinases
14.
Braz. j. med. biol. res ; 46(9): 746-751, 19/set. 2013. graf
Article in English | LILACS | ID: lil-686569

ABSTRACT

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.


Subject(s)
Animals , Mice , Hydrogen Peroxide/pharmacology , Intramolecular Oxidoreductases/drug effects , Macrophage Migration-Inhibitory Factors/drug effects , Myocytes, Cardiac/metabolism , Oxidants/pharmacology , Protein Kinase C/metabolism , src-Family Kinases/metabolism , Angiotensin II/metabolism , Blotting, Western , Cell Line , Immunohistochemistry , Intramolecular Oxidoreductases/genetics , Microscopy, Confocal , Macrophage Migration-Inhibitory Factors/genetics , Oxidative Stress/physiology , Protein Kinase Inhibitors/pharmacology , Real-Time Polymerase Chain Reaction , Renin-Angiotensin System/physiology
15.
Protein & Cell ; (12): 186-196, 2013.
Article in English | WPRIM | ID: wpr-757822

ABSTRACT

Studies on chronic myeloid leukemia (CML) have served as a paradigm for cancer research and therapy. These studies involve the identification of the first cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It becomes clear that leukemia stem cells (LSCs) in CML which are resistant to TKIs, and eradication of LSCs appears to be extremely difficult. Therefore, one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies. Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis, in which a rare stem cell population with limitless self-renewal potential gives rise to progenies that lack such potential. LSCs also possess biological features that are different from those of normal hematopoietic stem cells (HSCs) and are critical for their malignant characteristics. In this review, we summarize the latest progress in CML field, and attempt to understand the molecular mechanisms of survival regulation of LSCs.


Subject(s)
Animals , Humans , DNA-Binding Proteins , Genetics , Metabolism , Fusion Proteins, bcr-abl , Metabolism , Hypoxia-Inducible Factor 1 , Metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Drug Therapy , Metabolism , Pathology , Lipid Metabolism , Neoplastic Stem Cells , Metabolism , Protein Kinase Inhibitors , Pharmacology , Therapeutic Uses , Proto-Oncogene Proteins c-bcl-6 , src-Family Kinases , Metabolism
16.
Journal of Zhejiang University. Medical sciences ; (6): 538-542, 2013.
Article in Chinese | WPRIM | ID: wpr-252593

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of Src kinase inhibitor PP2 on intercellular communication of gap junction in breast cancer cells.</p><p><b>METHODS</b>Cultured breast cancer Hs578T cells were treated with various concentrations of pp2 (0,1,2,4,8,16,32 μmol/L) for 24h. Cell growth was determined by MTT assay; dye spread in Hs578T cells was measured by Parachute assay; and the expression of Src kinase in Hs578T cells was detected by Western blot.</p><p><b>RESULTS</b>MTT assay showed that the survive rate of Hs578T cells treated with PP2 (1 ≊ 8 μmol/L) was 98% ± 3% ≊ 94 % ± 4%. Parachute assay showed that compared to control group the standard normalized dye spread rates of Hs578T cells treated with 1,2,4 and 8 μmol/L PP2 were 1.60 ± 0.08,2.00 ± 0.05,2.20 ± 0.05 and 2.70 ± 0.09,respectively (all P<0.01). Moreover,compared to control group at the same time points,the standard normalized dye spread of Hs578T cells treated with 8 μmol/L PP2 for 6,12 and 24 h were 1.4 ± 0.05,1.7 ± 0.06,and 2.2 ± 0.07,respectively (all P<0.01). Western blot showed that the expression ratios of Src kinase/β-actin of Hs578T cells treated with 1,2,4 and 8 μmol/L PP2 for 24 h were 0.93 ± 0.02,0.70 ± 0.09,0.66 ± 0.09 and 0.36 ± 0.10,which were significantly inhibited compared with control group (P<0.05 or 0.01). And the expression ratio of Src kinase/β-actin of Hs578T cells treated with 8 μmol/L PP2 for 6,12 and 24h was 0.82 ± 0.03,0.66 ± 0.08 and 0.59 ±0.09, which were all inhibited significantly compared to control group (P<0.01).</p><p><b>CONCLUSION</b>PP2 enhances the gap junction function in breast cancer Hs578T cells, which is probably related to the inhibition of Src kinase.</p>


Subject(s)
Female , Humans , Breast Neoplasms , Pathology , Cell Line, Tumor , Gap Junctions , Pyrimidines , Pharmacology , src-Family Kinases , Metabolism
17.
Acta Pharmaceutica Sinica ; (12): 866-873, 2013.
Article in Chinese | WPRIM | ID: wpr-259538

ABSTRACT

Annexin A1 (ANXA1) is a kind of endogenous scaffold protein. Previous research showed that ANXA1 could increase markedly with multiple increase of drug resistance in K562/imatinib cell lines in vitro. Here the stable transfection cell strains K562-pEGFP-N1 which was the native control and K562-pEGFP-N1-ANXA1 which can stably express ANXA1 were established using the Lipofectamine 2000 in order to find whether ANXA1 involved in the drug resistance. Cell growth inhibition experiment via MTT and cell proliferation experiment via MTS showed that K562-pEGFP-N1-ANXA1 cell strain was more sensitive to imatinib than the K562-pEGFP-N1 cell strain, and however the ability of proliferation of K562-pEGFP-N1-ANXA1 cell strain did not change compared with the negative control. Western blotting results showed that the expression of proteins in Annexin family did not change; drug resistance proteins, Bcr-Abl/p-Bcr-Abl (Tyr245), Src family kinase for example, did not change; proteins related with cell proliferation and cell cycle, such as ERK1/2MAPK, p-38MAPK, CDK1 and Wee 1, did not change either in the K562-pEGFP-N1-ANXA1 cell strain compared with the negative control. The co-immunoprecipitation result showed that the interaction between ANXA1 and beta-actin in the K562-pEGFP-N1-ANXA1 cell strain increased markedly. The deduction was that ANXA1 may make the K562-pEGFP-N1-ANXA1 cell strain more sensitive to imatinib due to the increased uptake of imatinib via the increase of ANXA1 and the interaction between ANXA1 and beta-actin in the K562-pEGFP-N1-ANXA1 cell strain in vitro.


Subject(s)
Humans , Actins , Metabolism , Annexin A1 , Genetics , Metabolism , Antineoplastic Agents , Pharmacology , Cell Proliferation , Drug Resistance, Neoplasm , Fusion Proteins, bcr-abl , Metabolism , Imatinib Mesylate , Pharmacology , Immunoprecipitation , K562 Cells , Mitogen-Activated Protein Kinase 3 , Metabolism , Transfection , src-Family Kinases , Metabolism
18.
Cancer Research and Treatment ; : 126-133, 2013.
Article in English | WPRIM | ID: wpr-97205

ABSTRACT

PURPOSE: Leukemic promyelocytes have the unique ability to undergo differentiation after exposure to retinoic acid and both differentiation and apoptosis after exposure to arsenic trioxide (ATO). Recent studies have shown that inhibition of Src family kinases (SFKs) resulted in enhancement of retinoic acid-induced myeloid differentiation. MATERIALS AND METHODS: In this study, we investigated the question of whether the SFK inhibitor PP2 enhanced the differentiation of NB4 cells when combined with ATO as well as when combined with all-trans-retinoic acid (ATRA). In addition, we attempted to determine the difference in retinoic acid-induced gene expression between cells treated with PP2 in combination with ATRA and in combination with ATO. RESULTS: SFK inhibitor PP2 induced significant enhancement of ATRA- or ATO-induced differentiation of NB4 cells. A significantly stronger synergistic effect was observed when PP2 was combined with ATRA than when combined with ATO. Flow cytometric analysis demonstrated a significant increase in CD11b-positive granulocytes up to 60.73% and 31.58%, respectively. These results were confirmed by nitroblue tetrazolium staining. These effects were not related to apoptosis. Results of Annexin-V-fluorescein staining revealed that PP2 combined with ATRA or PP2 combined with ATO did not induce apoptosis in NB4 cells. Retinoic acid-induced gene expression was different in both groups. Intercellular adhesion molecule-1 expression showed a significant increase in cells treated with PP2 in combination with ATRA, whereas cathepsin D expression showed a significant increase in cells treated with PP2 in combination with ATO. CONCLUSION: Our data showed that SFK inhibitor PP2 enhanced acute promyelocytic leukemia cell differentiation when combined with either ATRA or ATO with difference in activation of retinoic acid-induced genes.


Subject(s)
Humans , Apoptosis , Arsenic , Arsenicals , Cathepsin D , Cell Differentiation , Cell Line , Gene Expression , Granulocyte Precursor Cells , Granulocytes , Intercellular Adhesion Molecule-1 , Leukemia, Promyelocytic, Acute , Nitroblue Tetrazolium , Oxides , Phosphotransferases , Pyrimidines , src-Family Kinases , Tretinoin
19.
Experimental & Molecular Medicine ; : e67-2013.
Article in English | WPRIM | ID: wpr-83998

ABSTRACT

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, alpha-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.


Subject(s)
Animals , Male , Rats , 4-Aminopyridine/pharmacology , Action Potentials , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Cells, Cultured , Ketanserin/pharmacology , Mesenteric Arteries/drug effects , Muscle Contraction , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/drug effects , Nifedipine/pharmacology , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Rats, Sprague-Dawley , Receptor, Serotonin, 5-HT2A/metabolism , Serotonin/pharmacology , Serotonin 5-HT2 Receptor Antagonists/pharmacology , Spiperone/pharmacology , Vasoconstriction , src-Family Kinases/antagonists & inhibitors
20.
Chinese Journal of Cancer ; (12): 75-83, 2013.
Article in English | WPRIM | ID: wpr-295829

ABSTRACT

Protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process.


Subject(s)
Humans , Cell Adhesion , Cell Movement , Focal Adhesion Protein-Tyrosine Kinases , Metabolism , Neoplasms , Metabolism , Pathology , Oxidation-Reduction , Phosphorylation , Protein Processing, Post-Translational , Protein Tyrosine Phosphatase, Non-Receptor Type 12 , Metabolism , Shc Signaling Adaptor Proteins , Metabolism , rho GTP-Binding Proteins , Metabolism , src-Family Kinases , Metabolism
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