Src/RUNX2 Modulates the Migration of Vascular Smooth Muscle Cells Induced by Cyclic Stretch / 医用生物力学

Objective To investigate the effect of cyclic stretch on Src and Runt-related transcription factor 2 (RUNX2), and their pivotal roles in migration of vascular smooth muscle cells (VSMCs). Methods The 5% cyclic stretch (to simulate normotensive physiological condition) or 15% cyclic stretch (to simulate hypertensive pathological condition) was applied to VSMCs by FX-5000T system. Western blotting was used to detect the expression of RUNX2 and phosphorylation of Src in VSMCs. The Ingenuity Pathway Analysis (IPA) bioinformatic software was used to analyze the potential regulatory effect of Src on RUNX2. Small interfering RNA (siRNA) was transfected to decrease the expression of RUNX2. Src inhibitor-1 was used to repress Src kinase activity; Wound-healing assay was applied to detect VSMC migration. Results Compared with 5% cyclic stretch, 15% cyclic stretch significantly increased RUNX2 expression in VSMCs. Under both static and 15% cyclic stretch conditions, VSMC migration was significantly inhibited after reducing RUNX2 expression with siRNA transfection. IPA indicated that Src kinase might be the upstream modulator of RUNX2, and Western blotting validated that RUNX2 expression was significantly decreased after inhibiting Src. Furthermore, under 15% cyclic stretch, Src inhibitor-1 markedly repressed RUNX2 expression and VSMC migration.Conclusions High cyclic stretch increased phosphorylation of Src kinase and expression of RUNX2, which subsequently induced VSMC abnormal migration. Exploring the mechanobiological mechanism of VSMC migration regulated by cyclic stretch may contribute to further revealing the mechanism of vascular physiological homeostasis and vascular pathological remodeling, as well as providing new perspective for the translational research of vascular remodeling upon hypertension.

Piezol involved in electrical remodeling in atrial myocytes induced by high hydrostatic pressure through Src kinase / 中国药理学通报

Aim To investigate the role of mechano- sensitive ion channel Piezol in regulating electrical re-modeling of atrial myocytes induced by hypertension and to further explore the potential mechanisms.Methods Spontaneously hypertensive rats ( SHR ) aged 30 - 32 weeks treated with or without valsartan (30 mg • kg 1 • d 1 ) were used.Wistar rats were used as control.Western blot was used to detect the protein expression of Piezol , Src and Cavl.2 in atrial appendages of rats and in atrial myocytes ( HL-1 cells) exposed to different levels of high hydrostatic pressure (20 and 40 mmHg) , Piezol inhibitor (GsmTx4) and agonist ( Yodal ) in vitro.Whole-cell patch clamp technique was employed to detect L-tvpe calcium current (ICa, ) and action potential duration ( APD) of atrial myocytes.Results Compared with Wistar rats in control group, the protein expressions of Piezol and Src significantly increased and the expression of Cavl.2 decreased in SHR group (P < 0.05 ), while the a- bove changes could he reversed in SHR treated with valsartan( P < 0.05 ) .Meanwhile, higher hydrostatic pressure (40 mniHg) could increase the expressions of Piezol and Src in HL-1 cells( P <0.05) and decrease the protein expression of Cavl.2 (P <0.05 ) , accompanied by a shortened APD and a decreased ICa,.GsmTx4 could significantly reverse the above changes.In addition, Piezol agonist Yodal could simulate electrical remodeling and related signal molecule changes in atrial myocytes induced by the high hydrostatic pressure.Conclusions Mechanosensitive ion channel Piezol participates in electrical remodeling induced by hypertension via activating Src kinase signaling pathway and then leading to the decrease of ICa ,.

Involvement of Src kinase in the down-regulation of ultra-rapid delayed rectifier K(+)current induced by tumor necrosis factor-α in cardiomyocytes / 中华心血管病杂志

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.

Inhibition of invasion and metastasis of human tonguesquamous cell carcinoma Tca8113 cells by Src kinase inhibitor PP2 / 西安交通大学学报(医学版)

Objective: To investigate the inhibitory effects of Src kinase inhibitor PP2 on migration and invasion of Tca8113 cells. Methods: Tca8113 cells were cultured for 24 h with 5 mol/L, 10 mol/L and 20 micron mol/L of Src kinase inhibitor PP2. The effects of PP2 on the invasion and migration of Tca8113 cells were measured with Transwell chamber and scratch method, respectively. Results: After the treatment with PP2 for 24 h, the expression of p-Src in 5, 10, 20 μmol/L of Src kinase inhibitor PP2 treatment groups was significantly lower than that of the non-drug treatment group (all P<0.05). The number of Tca8113 cells in the non-drug treatment group and the 5, 10, and 20 μmol/L of Src kinase inhibitor PP2 treatment groups was (232.76±28.65), (186.53±21.34), (129.18±17.96), and (37.82±12.41), respectively; the number of migratory cells was (259.38±25.27), (193.45±20.18), (143.24±18.04), and (32.94±14.39), respectively, the cell migration rate was (11.51±0.84)%, (8.06±0.51)%, (5.13±0.57)%, and (3.18±0.12)%, respectively; the overall difference was statistically significant (F=73.852, 85.687, 48.157, all P=0.000). It had a negative correlation with PP2 dose. Conclusion: Src kinase inhibitor PP2 can effectively inhibit the invasion and migration of Tca8113 cells in the concentration-dependent manner, and it may have certain clinical value in the treatment of human tongue squamous cell carcinoma.

SRC kinase inhibitor PP2 inhibits invasion and metastasis of lung cancer A549 cells by upregulating connexin43 expression / 南方医科大学学报

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.

El hexaclorobenceno como factor de riesgo en el cáncer de mama / Hexachlorobenzene as a risk factor in breast cancer

Hexachlorobenzene (HCB) is a widespread environmental pollutant and an endocrine disruptor. Chronic exposure of humans to HCB elicits porphyria, neurologic symptoms, immune disorders and thyroid dysfunctions. It is a dioxin-like compound and a weak ligand of the AhR (aryl hydrocarbon receptor), a transcription factor that modulates genes related to detoxification, proliferation, migration and invasion. This study was carried out to revise the results of HCB action on mammary gland and breast cancer, summarizing the main ideas of its mechanism of action. HCB increases tumor development and active c-Src/EGFR (epidermal growth factor receptor) signaling pathways, while reducing tyrosine537-ER-alpha (estrogen receptor-alpha) phosphorylation, and promoting a phenotype with enhanced malignancy and lung metastasis in different animal models. In a rat mammary gland, HCB promotes an estrogenic microenvironment by activation of ER-alpha and Insulin/IGFs (insulin growth factors) pathways. HCB induces cell proliferation, promoting cell cycle progression and enhancing cyclin D1 expression and c-Src/p27 interaction in (ER-alpha) MCF-7 human breast cancer cell line. In (ER-alpha)(-) MDA-MB-231 breast cancer cells, the pesticide enhances cell migration and invasion as well as metalloproteases and TGF-beta1 (transformig growth factor-beta1) expression. In conclusion our current study suggests that alterations in the estrogenic microenvironment may influence the biological behavior of mammary gland or breast tumors, leading to preneoplastic lesions or enhanced malignancy, respectively. Our findings suggest that HCB may be a risk factor for human breast cancer progression.

El hexaclorobenceno (HCB) es un contaminante ambiental ampliamente distribuido y un desorganizador endocrino. Su exposición crónica en seres humanos produce porfiria, síntomas neurológicos, trastornos inmunitarios y disfunciones tiroideas. Es un agonista débil del receptor de hidrocarburos aromáticos (AhR), un factor de transcripción que modula genes relacionados con el metabolismo de xenobióticos, la proliferación, la migración y la invasión. Nuestro objetivo es revisar los efectos del HCB en la glándula mamaria y el cáncer mamario, resumiendo los principales mecanismos de acción. El HCB aumenta el desarrollo tumoral y activa vías de señalización de c-Src/receptor del factor de crecimiento epidérmico (EGFR), mientras que disminuye la fosforilación de tirosina 537/receptor de estrógenos alfa (RE-alfa), promoviendo un fenotipo de mayor malignidad y metástasis pulmonar en diferentes modelos con animales. En la glándula mamaria de rata genera un microambiente estrogénico por activación del RE-alfa y las vías de insulina/factores de crecimiento similares a la insulina (IGF). En células de cáncer mamario humanas MCF-7 (RE-alfa) induce proliferación celular, promoviendo la progresión del ciclo, aumentando la ciclina D1 y la interacción p27/c-Src. En MDA-MB-231 (-RE-alfa) estimula la migración e invasión, así como la expresión de metaloproteasas y factor de crecimiento transformante beta 1 (TGF-beta 1). Estos estudios indican que las alteraciones en el microambiente estrogénico podrían influir el comportamiento biológico de la glándula mamaria y los tumores, lo que provoca lesiones preneoplásicas o aumento en la malignidad tumoral mamaria. Nuestros hallazgos sugieren que el HCB podría ser un factor de riesgo para la progresión del cáncer de mama humano.

Activación de la célula T, alteraciones en el lupus eritematoso sistémico, una revisión narrativa / T-cell activation, alterations in systemic lupus erythematosus: A narrative review

Resumen La activación de los linfocitos T se inicia a través de la presentación de antígenos endógenos o exógenos por células presentadoras de antígenos a través del complejo mayor de histocompatibilidad, el cual se une a un receptor especializado presente en los linfocitos T. Este reconocimiento desencadena una cascada de señalización intracelular que conlleva a un aumento en la expresión de integrinas, modificaciones del citoesqueleto y producción de factores de transcripción involucrados en la liberación de citocinas y mediadores inflamatorios. Uno de los inductores más importantes en la activación celular es el complejo enzimático con acción tirosina cinasa. Las cinasas que pertenecen a la familia SRC (SFK), FYN y LCK están involucradas en un gran número de procesos importantes en la activación, modulación de la respuesta linfocitaria y el desarrollo de enfermedades autoinmunes. La regulación de la señalización de las cinasas, así como de proteínas adaptadoras involucradas en la activación del linfocito T, son fundamentales para mantener el umbral de activación y modulación de la respuesta del linfocito. La fosforilación de sitios de regulación positiva de estas proteínas es importante para permitir una configuración activa de la proteína y de esta forma su máxima capacidad como cinasa. La fosforilación de los sitios de regulación negativa conlleva a una configuración cerrada de la proteína de tal forma que reduce su función de cinasa e inhibe su función. Las alteraciones en la señalización por modificación de algunas proteínas citoplasmáticas se asocian en algunos casos al desarrollo de enfermedades autoinmunes, como el lupus eritematoso sistémico. En condiciones fisiológicas, el complejo receptor de linfocitos T se reagrupa con complejos proteicos que interactúan armónicamente para generar una sen al interna. Los eventos de señalización alterados son en parte los responsables de una expresión anómala de citocinas, entre ellas la interleucina-6 (IL-6), IL-10, IL-2, IFN y CD40 ligando; estas modificaciones alteran la capacidad de los linfocitos T para sobre estimular a los linfocitos B, traduciéndose en un aumento en la producción de autoanticuerpos y en el desencadenamiento de la enfermedad autoinmune.

Abstract The activation of T cells is initiated by the presentation of exogenous or endogenous antigens, by antigen presenting cells through the major histocompatibility complex, which binds to a special receptor on T cells. This acknowledgement triggers a cascade of intracellular signalling that leads to an increase in integrin expression, cytoskeletal modifications, and transcription factors production involved in the liberation of cytokines and inflammatory mediators. One of the most important inducers in cell activation is the enzymatic complex with tyrosine kinase action. The kinases which belong to the SRC (SFK) LCK and FYN family have been involved in a large number of important processes in the activation and modulation of the T cells response, as well as in the development of autoimmune diseases. Regulating the kinases signalling, as well as the adapter proteins involved in T cell activation, is essential for maintaining an activation threshold, as well as the modulation of cell response. The phosphorylation of the positive regulation sites of these proteins is important to allow an active configuration of the protein and thereby its maximum capacity as kinase. The phosphorylation of negative regulation sites leads to a closed configuration of the protein that reduces its kinase function, and thereby inhibits its own function. The alteration in signalling by the modification of certain cytoplasmic proteins in some cases is associated with the development of autoimmune diseases, such as systemic lupus erythematosus. Under physiological conditions the T cell receptor complex regroups with protein complexes that interact harmonically to generate an internal signal. The altered signalling events are partly responsible for an anomalous expression of cytokines, including the interleukin-6 (IL-6), IL-10, IL-2, IFN, and CD40 linking, these modifications affects the cells ability to over-stimulate T and B cells, resulting in an increased production of autoantibodies and the triggering of the autoimmune disease.

Inhibiting effects of Src kinase inhibitor on TGF-β1 induced epithelial-mesenchymal transition of human RPE cells / 中华实验眼科杂志

Objective To investigate the inhibiting effect of CGP77675 (CGP),a Src inhibitor,on epithelial-mesenchymal transition (EMT) of human retinal pigment epithelial (RPE) cells induced by transformation growth factor-β1 (TGF-β1).Methods Human RPE cell line (ARPE19 cells) was cultured in vitro and divided into control group,TGF-β1 group and TGF-β1 +CGP group.Corresponding agent was added into culture medium based on grouping.The morphology of the cells were examined under the optical microscope 3 days after culture.The expressions of EMT-related genes and proteins in the cells were detected by real-time quantitative PCR and Western blot,respectively,including fibronectin 1 (FN 1),and plasminogen activation inhibitor 1 (PAI1),and the expressions of zonula occludens protein 1 (ZO1) and cytoskeleton protein filamentous actin (F-actin) were detected by immunofluorescence staining.MTT assay was employed to evaluate the cell proliferation rate.The migration distance of the cells was measured by scratch test.Results The ARPE19 cells in the control group showed an epithelial-like morphology and F-actin and ZO-1 were expressed along cell membrane.In the TGF-β1 group,the cells appeared to be fibrous-like,and the fluorescence staining of F-actin was disordered and ZO-1 was discontinuous on the cell membrane.The cells in the TGF-β1 +CGP group remained to be an epithelial-like in shape with clear and complete expressions of F-actin and ZO-1.The relative expressions of FN1 mRNA and PAI1 mRNA in the cells were 0.211 ± 0.080 and 0.116±0.073,1.000±0.001 and 1.000±0.001,0.368±0.097 and 0.362±0.048 in the control group,TGF-β1 group and TGF-β1 +CGP groups,showing significant differences among the groups (F=33.14,82.92;both at P＜0.01),with the highest expressions ofFN1 mRNA and PAI1 mRNA in the TGF-β1 group (all at P＜0.05).The relative expressions of FN1 and PAI1 proteins were 0.166±0.055 and 0.327±0.066,1.000±0.001 and 1.000± 0.001,0.143 ± 0.030 and 0.260 ± 0.077 in the control group,TGF-β1 group and TGF-β1 + CGP group,with significant differences among three groups (F=181.90,48.85;both at P＜0.01),and the expressions FN1 and PAI1 proteins were significantly higher in the TGF-β1 than those in the control group and TGF-β1 +CGP group (all at P＜0.05).The cell proliferative rate in the TGF-β1+CGP group was (79.30±3.44) ％ and (54.80±7.39) ％ at the third day and seventh day after culture,which were significantly reduced in comparison with (99.50 ± 1.00)％ and (99.10±0.50)％ in the control group as well as (95.10±4.20)％ and (92.10±4.50)％ in the TGF-β1 group (all at P＜0.05).The migration distance was disappeared in the TGF-β1 group,and the scratch width was not obviously changed in the TGF-β1 +CGP group.Conclusions Src inhibitor can inhibit EMT process of ARPE19 cells induced by TGF-β1,indicating that Src signaling pathway may play a critical role in EMT of RPE cells.

Advances of Src kinase family and paclitaxel resistance / 中国药科大学学报

Src family kinase (SFK) highly expresses in many types of cancers,broadly adjusting their malignant behaviors.Paclitaxel is a widely used chemical agent.However,because of constant resistance,the effect of paclitaxel has been greatly attenuated.The present review summaries the recent research progress of the structure and adjustment of SFK and the molecular mechanism of paclitaxel resistance,as well as the regulation of SFK on paclitaxel resistance,in order to provide new references and evidences upon the paclitaxel-based tumor therapy.

Expression of p-FRK and its prognostic analysis in non-small cell lung cancer patients / 临床与实验病理学杂志

Purpose To investigate the expression of pFRK protein in non-small cell lung cancer (NSCLC) patients and to explore its prognostic value.Methods The expression of p-FRK protein in tumor tissues and corresponding adjacent normal pulmonary tissues from 162 NSCLC patients was detected by immunohistochemistry of EnVision two-step.The correlation among p-FRK expression,age,gender,clinicopathologic features,pTNM stage and metastasis of NSCLC patients was also analyzed.whether p-FRK could be used as an independent predictor of prognosis for patients with NSCLC was further determined.Results The positive expression rate of p-FRK in NSCLC tissues (51.9％) was significantly higher than that of adjacent normal lung tissues (11.7％) (P =0.001).The expression of p-FRK in NSCLC was significantly correlated with histopathologic type,lymph node status and pTNM stage (P ＜0.05).Kaplan-Meier survival analysis showed that there was a negative correlation between p-FRK positive rate and postoperative overall survival (x2 =17.849,P ＜0.001).Multivariate analysis showed that p-FRK expression was an independent prognostic factor for overall survival of NSCLC patients (HR =0.496,95％ CI:0.295-0.836,P ＜ 0.05).Conclusion As a novel NSCLC biomarker,the expression of p-FRK may predict a poor prognosis in the patients with NSCLC.

Antitumor Effect of KX-01 through Inhibiting Src Family Kinases and Mitosis / Journal of the Korean Cancer Association, 대한암학회지

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.

Effect of over expression of C-terminal Src kinase binding protein (Cbp) and Cbp palmitoylation to proliferation of T-leukemia Jurkat cell in nude mice / 中国免疫学杂志

Objective:To establish T lineage leukemia Jurkat cell mice model of over expression of C-terminal Src kinase binding protein( Cbp ) and Cbp palmitoylation and to research the effect of Cbp and Cbp palmitoylation to proliferation of Jurkat cell.Methods:Virus transfected cell of neg-EGFP,Cbp-EGFP and Cbp-m-EGFP were used in mice model.24 female BALB/c-nu mice were randomly divided into blank control group,empty virus control group,over expression of CBP group and Cbp palmitoylation group, 6 mice in each group.The nude mice were weighed in 0,1,2,3,4,5 weeks.The amount of white blood cell in peripheral blood were counted in 0, 1, 2, 3, 4, 5 weeks.The proliferation of Jurkat cell in peripheral blood of mice were observed by laser confocal microscope.The pathological changes of liver were observed using HE staining.The proliferation of Jurkat cell in the bone marrow and peripheral blood of mice were detected with flow cytometry.Results:The weight of mice in over expression of Cbp group was less than that in blank control group,but higher than that in empty virus control group and Cbp palmitoylation group.The weight of mice in Cbp palmitoylation group was less than that in blank control group,empty virus control group and over expression of Cbp group.The amount of white blood cell in peripheral blood and proliferation of Jurkat cell in liver, bone marrow and peripheral blood of mice in over expression of Cbp group was higher than that in blank control group, but less than that in empty virus control group and Cbp palmitoylation group.The amount of white blood cell in peripheral blood and proliferation of Jurkat cell in liver, bone marrow and peripheral blood of mice in Cbp palmitoylation group was higher than that in blank control group,empty virus control group and over ex-pression of Cbp group.Conclusion:Over expression of Cbp and Cbp palmitoylation in T lineage leukemia Jurkat cell mice model was established.Over expression of Cbp has inhibitory effect on the proliferation of Jurkat cell.Cbp palmitoylation has promotable effect on the proliferation of Jurkat cell.

Mechanism study of dasatinib inhibiting phosphorylation of androgen receptor in prostatic cancer cells / 肿瘤研究与临床

Objective To investigate the mechanism of dasatinib,tyrosine kinase inhibitor,inhibiting androgen receptor (AR) phosphorylation in prostate cancer cells.Methods HEK-293T and COS7 cell lines were cotransfected by wild-type (WT)-AR,ARY267F or ARY534F with Ack1 or Src,respectively,and Western blot was used to detect the AR phosphorylation sites.LNCaP cells were treated by EGF or heregulin without androgen,then Western blot was used to detect AR phosphorylation.After these LNCaP cells were treated by dasatinib or transfection with siRNA to silence Ack1 or Src gene,Western blot was used to observe the effect on AR phosphorylation,and quantitative real-time reverse transcription polymerase chain (RT-PCR)was applied to detect PSA mRNA and hk2 mRNA.Results After transfection,Ack1 kinase mediated the phosphorylation of AR Tyr267 in HEK-293T cells,and Src mediated AR Tyr534 phosphorylation in COS7cells.When LNCaP cells were treated by heregulin,AR Tyr267 was phosphorylated,but its phosphorylation was inhibited after these cells were treated by dasatinib or ack1 gene was silenced.When LNCaP cells were treated by EGF,AR Tyr534 was phosphorylated,but its phosphorylation was inhibited after these cells were treated by dasatinib or Src gene was silenced.EGF or heregulin raised endogenous AR target gene,PSA and hK2,mRNA levels in LNCaP cells (P ＜ 0.05).However,after these cells were treated by dasatinib,PSA and hK2 mRNA levels induced by heregulin were decreased (P ＜ 0.05),but those induced by EGF PSA were no significant changes (P ＞ 0.05).Conclusion Dasatinib can inhibit AR Tyr267 and AR Tyr 534phosphorylation,and it may play a significant role in anti-prostate cancer cells by inhibiting Ack1-mediated AR Tyr-267 phosphorylation and the expression of PSA mRNA and hk2 mRNA induced by heregulin.

Hepatitis B virus X protein induces epithelial-mesenchymal transition through c-Src activation in liver cancer cells / 中华肝胆外科杂志

Objective To examine the role of c-Src activation in hepatitis B virus X (HBx) protein induced epithelial-mesenchymal transition (EMT) in liver cancer.Methods SMMC-7721 liver cancer cells were transfected with HBx gene to induce EMT and the activated c-Src expression was evaluated by Western blot.Both the morphological changes and the epithelial and mesenchymal markers expression (real-time PCR,western blot and immunocytochemistry) of HBx-transfected SMMC-7721 cell treated by c-Src kinase inhibitor PP2 and negative control PP3 were observed and compared,respectively.Results The activated c-Src expression in HBx gene transfected SMMC-7721 cells was significantly increased compared to that in mock transfected cells,c-Src kinase inhibitor PP2 could enable the HBx-transfected SMMC-7721 cells to transmit from spindle-like shape to original epithelial morphology.Western blot and immunocytochemistry confirmed that the expression of epithelial markers and mesenchymal markers almost returned to the levels of parental cells,indicating the mesenchymal-epithelial transition.Conclusions c-Src activation plays a key role in the process of EMT induced by HBx protein in SMMC-7721 cells.

The impact of α-particles irradiation on src kinase activity and autophagy system mediated by ROS / 中华放射医学与防护杂志

Objective To investigate the modulation role of autophagy in radiation-induced cell death by detecting the response of src kinase activity and autophagy in HEK293 cells irradiated with different dose of α-particle.Methods HEK293 cells were irradiated by contral group (0 cGy) a low dose group (10 cGy) and high dose group (300 cGy) α-particles.Molecular probe 2',7'-dichlorofluorescin (DCFHDA) was used to detect the cell ROS.The src kinase activity and endogenous protein level of LC3Ⅰ/Ⅱ were monitored by Western Blot.Cell death rate of irradiated cells pretreated with autophagy inducer of rapamycin was tested by flow cytometry.Results Compared with control group,the ratio of LC3Ⅰ/Ⅱ decreased (t =4.07,P ＜ 0.05) and the percentage of cells with GFP-LC3 punctuate dots increased (t =12.29,P ＜0.05) under 10 cGy irradiation,indicating the induction of autophagy.On the contrary,the ratio of LC3Ⅰ/Ⅱ increased (t =2.93,P ＜ 0.05) and the GFP-LC3 morphology had no obvious change under 300 cGy irradiation.The cellular ROS level reached to the maximum value at 4 h postirradiation.Both 10 cGy and 300 cGy irradiation could elevate the ROS level (t =17.93,22.88,P ＜0.05),whereas the amplitude of elevation of 300 cGy irradiation was higher than that of 10 cGy irradiation (t =15.76,22.66,14.22,P ＜ 0.05).Compared with control group,the 419th site of tyrosine residue in src kinase manifested hyper-phosphorylation (t =5.66,P ＜0.05) under 10 cGy irradiation whereas it had hypo-phosphorylation under 300 cGy irradiation (t =4.67,P ＜ 0.05).Treatment of cells with DMSO could partly restore the impact on src kinase activity under high or low dose irradiation.Pre-treating the cells with autophagy inducer rapamycin could reduce cell death under 300 cGy irradiations (t =12.14,P ＜ 0.05).Conclusions High or low dose of α-particles irradiation could inhibit or activate src kinase and autophagy system,respectively.ROS mediated the response of src kinase activity and autophagy system induced by irradiation.Modulation of autophagy could desensitize cell responses to irradiation.

Effects of tyrosine kinase Src during C.albicans infection in murine macrophage / 实用医学杂志

Objective To investigate the role of tyrosine kinase Src in a murine C.albicans infection model. Methods Observed cell proliferation by alarmarblue assay at 2, 24 and 48 h after Src inhibitor PP2 treatment. Phagocytosis was determined by a fluorometric assay. Cytokine TNF-αand IL-10 production was detected by ELISA. Results The 0~33.3 μmol/L PP2 had no effect on cell proliferation after PP2 treatment for 2 h. When the PP2 treatment extended to 24 or 48 h, PP2 (11.1, 33.3μmol/L) showed significant inhibition on cell proliferation with 78%, 9%, and 54%,13%, respectively. At 48 h after 11.1μmol/L PP2 treatment, the internalization of C.albicans in macrophage is significantly inhibited, contributing to the inhibition of cell proliferation. However, the 11.1 and 33.3μmol/L PP2 significantly inhibited the cytokine TNF-αand IL-10 production during C.albicans infection (P<0.01). Conclusion Src kinase played an important role during C.albicans infection, especially for the cytokine TNF-αand IL-10 production.

Osteoporosis: Current Management Guidelines.

Osteoporosis is a multifactorial progressive skeletal disorder characterized by reduced bone mass and deterioration of bone microarchitecture. Fragility fractures, the consequence of osteoporosis, are responsible for excess mortality, morbidity, chronic pain, admission to hospitals and economic costs. Approximately 1.6 million hip fractures occur each year worldwide and the incidence is set to increase to 6.3 million by 2050. Preventive measures should be started at an early age and should include smoking cessation and weight-bearing exercises. Pharmacologic prevention methods include calcium supplementation and administration of raloxifene or bisphosphonates. No treatment can completely reverse established osteoporosis. Early intervention can prevent osteoporosis in most people. For patients with established osteoporosis, medical intervention can halt its progression. Currently available therapies include bisphosphonates, selective estrogen receptor modulators (SERMs), hormone replacement therapy (HRT), denosumab, teriparatide, calcitonin and strontium ranelate. Cathepsin K inhibitors (balicatib and odanacatib) are among recent drugs under development. Saracatinib, a novel orally available competitive inhibitor of Src kinase has been shown to inhibit bone resorption in vitro. Lasofoxifene, bazedoxifene and arzoxifene are new SERMs in late-stage treatment trials. Nonpharmacological measures are required when patients experience adverse effects because of drug therapy, when symptoms are not controlled by drug therapy alone or when patient is not willing to take drugs for a prolonged duration.

Effects of substrate stiffness on confluent growth of hepatic and hepatoma carcinoma cells / 医用生物力学

Objective To investigate the cause of differences in confluent growth between hepatic(L02) and hepatoma carcinoma(HCCLM3) cells by comparing responses of the two cells to different substrate stiffness (0.5, 4 kPa and glass). MethodsThe real-time photomicrography, immunofluorescence staining, flow cytometry, and Western Blotting techniques were respectively employed to observe the morphological characteristics, the cytoskeleton conformation and the distribution of E-cad of confluent L02 and HCCLM3 cells on different substrates, and test the changes in expression of E-cad, Integrinβ1 and p-Src. Results (1) Confluent L02 cells displayed a round or cubic shape, while HCCLM3 cells showed a polygon shape. The morphology of HCCLM3 cells were spread and polarized more obviously than that of L02 cells. With the increase of substrate stiffness, the variation of L02 cells with time was smaller than that of HCCLM3 cells. (2) The cytoskeleton of confluent L02 cells showed a ring-like conformation under the cortex, and E-cad was located at the cell-cell contact sites. However, the ring-like cytoskeleton of HCCLM3 cells was incomplete and distributed radially along the basement, while E-cad was dispersed in cytoplasm. (3) As the substrate stiffness increased, expression of E-cadherin in both L02 and HCCLM3 cells was significantly decreased (P<0.01), while the level of p-Src and integrinβ1 was increased significantly, with greater changes in HCCLM3 cells than in L02 cells. Conclusions The assembling of cortical ring-like cytoskeleton was positively correlated with the location of E-cad at the cell-cell contact sites. The substrate stiffness showed a more obvious impact on the balanced regulation between cadherin and integrin mediated adhesion system of hepatocarcinoma cells than that of hepatic cells.

Src Family Kinase Inhibitor PP2 Has Different Effects on All-Trans-Retinoic Acid or Arsenic Trioxide-Induced Differentiation of an Acute Promyelocytic Leukemia Cell Line / Journal of the Korean Cancer Association, 대한암학회지

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.

Src Kinase Regulates Nitric Oxide-induced Dedifferentiation and Cyclooxygenase-2 Expression in Articular Chondrocytes via p38 Kinase-dependent Pathway

BACKGROUND: Nitric oxide (NO) in articular chondrocytes regulates dedifferentiation and inflammatory responses by modulating MAP kinases. In this study, we investigated whether the Src kinase in chondrocytes regulates NO-induced dedifferentiation and cyclooxygenase-2 (COX-2) expression. METHODS: Primary chondrocytes were treated with various concentrations of SNP for 24 h. The COX-2 and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin E(2) (PGE(2)) was determined by using a PGE(2) assay kit. Expression and distribution of p-Caveolin and COX-2 in rabbit articular chondrocytes and cartilage explants were determined by immunohistochemical staining and immunocytochemical staining, respectively. RESULTS: SNP treatment stimulated Src kinase activation in a dose-dependent manner in articular chondrocytes. The Src kinase inhibitors PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine], a significantly blocked SNP-induced p38 kinase and caveolin-1 activation in a dose-dependent manner. Therefore, to determine whether Src kinase activation is associated with dedifferentiation and/or COX-2 expression and PGE(2) production. As expected, PP2 potentiated SNP-stimulated dedifferentiation, but completely blocked both COX-2 expression and PGE2 production. And also, levels of p-Caveolin and COX-2 protein expression were increased in SNP-treated primary chondrocytes and osteoarthritic and rheumatoid arthritic cartilage, suggesting that p-Caveolin may play a role in the inflammatory responses of arthritic cartilage. CONCLUSION: Our previously studies indicated that NO caused dedifferentiation and COX-2 expression is regulated by p38 kinase through caveolin-1 (1). Therefore, our results collectively suggest that Src kinase regulates NO-induced dedifferentiation and COX-2 expression in chondrocytes via p38 kinase in association with caveolin-1.