OBJECTIVESTo investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on integrin signaling and apoptosis in fibronectin (FN) -stimulated hepatic stellate cells (HSCs).

METHODS3H-thymidine incorporation, annexin-V/propidium iodide double-labeled flow cytometry (FCM) and transmission electron microscopy were employed to estimate the influence of RGDS on the proliferation and apoptosis of HSCs. And the adhesion rates were observed by toluidine blue colorimetric assay. The expression of focal adhesion kinase (FAK) mRNA and protein in HSCs was detected using RT-PCR and western blotting analysis, respectively.

RESULTSRGDS tetrapeptide at the concentrations of 25 microg/ml, 50 microg/ml and 100 microg/ml inhibited the proliferation of HSCs and induced HSCs apoptosis in dose-dependent and time-dependent manners, with the apoptotic rates of 9.49%, 27.67%, 31.59%, and the necrotic rates of 3.47%, 5.38%, 9.10%, respectively. Both the rates were higher than those in FN group (apoptotic rate: 3.44%; necrotic rate: 2.39%), F=8.02, P<0.05. After adding RGDS tetrapeptide to HSCs for 2 hours, the adhesive inhibition rates were 8.82%, 29.41% and 45.59%, respectively, which were higher than that in FN group (F=20.58, P<0.01). After exposure of HSCs to RGDS tetrapeptide for 24 hours, FAK protein decreased, and FAK mRNA was down-regulated earlier, about 2 hours after exposure to RGDS tetrapeptide.

CONCLUSIONThese results suggest that RGDS tetrapeptide may induce apoptosis of HSC in both dose-dependent and time-dependent manners in vitro, which may be related to the disruption of cell matrix adhesion and down-regulation of FAK expression.

Apoptosis ; drug effects ; Fibronectins ; pharmacology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Hepatocytes ; cytology ; physiology ; Humans ; Integrins ; metabolism ; physiology ; Liver ; cytology ; physiology ; Oligopeptides ; pharmacology ; Platelet Aggregation Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; biosynthesis ; genetics ; metabolism ; Signal Transduction

OBJECTIVEFMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1.

METHODSFLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h.

RESULTSFLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001.

CONCLUSIONThe suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.

Apoptosis ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Child ; Humans ; Leukemia, Monocytic, Acute ; enzymology ; pathology ; Protein-Tyrosine Kinases ; metabolism ; RNA Interference ; physiology ; RNA, Small Interfering ; pharmacology ; Receptor Protein-Tyrosine Kinases ; metabolism ; fms-Like Tyrosine Kinase 3 ; metabolism

The aim of the present study was to observe whether protein tyrosine kinase (PTK) within the nucleus tractus solitarius (NTS) was involved in the regulation of ventilatory responses of peripheral chemoreflex. The experiments were performed on anesthetized, immobilized and artificially ventilated rabbits. Peripheral chemoreflex was elicited by ventilating the animal with 10% O2-balance 90% N2. Changes in the peak amplitude and frequency of integrated phrenic nerve activity were observed. The ventilatory responses of peripheral chemoreflex following 0.1 microl microinjection within the NTS of either PTK inhibitor genistein (10 mol/L), AMPA glutamate receptor inhibitor CNQX (10 mol/L),or inactive PTK inhibitor daidzein (10 mol/L) were recorded. The results are as follows: Both genistein and CNQX attenuated the ventilatory responses of peripheral chemoreflex, while no changes occurred following daidzein. The amplitude of integrated phrenic nerve discharge and the phrenic burst frequency were decreased by (-21.77+/-6.93)% and (-24.70+/-7.61)% respectively after administration of genistein. CNQX resulted in similar decreases in the amplitude of phrenic nerve discharge (-27.13+/-7.63)% and the burst frequency (-21.34+/-4.88)%. In addition, the inhibitory effects of CNQX and genistein were the same whether they were applied alone or one after another, indicating that they had no cooperative effects. The results obtained suggest that PTK within the NTS regulates the peripheral chemoreflex control of respiration and that this regulation of PTK may be mediated through the phosphorylation of AMPA receptors in NTS neurons.
Animals ; Brain Stem ; enzymology ; physiology ; Chemoreceptor Cells ; physiology ; Female ; Male ; Protein-Tyrosine Kinases ; physiology ; Rabbits ; Receptors, AMPA ; physiology ; Respiration ; Solitary Nucleus ; enzymology

Cell migration plays an important role in repair of injury, angiogenesis, cancer metastasis and so on. In this paper the effects of basic fibroblast growth factor (bFGF) of different concentrations on ECV-304 cell migration, and the dynamic changes in focal adhesion kinase (FAK) were observed. The relationship between FAK and cell migration induced by bFGF was studied. A ECV-304 cell scratch wound model was established and the images of cell migration were quantitatively measured using a computer-assisted videomicroscopic system. The dynamic changes in FAK content (Western blot), FAK activity (immunoprecipitation plus Western blot) and FAK mRNA (RT-PCR) were measured in vitro. The expression of integrin alpha3 was investigated using immunocytochemical staining (ABC method). The results showed that bFGF produced a dual-phase regulatory effect on ECV-304 migration when the cell confluent areas reached 90%-95% in culture. It was found that compared with the control group (0 ng/ml bFGF), the cell migration was stimulated (P<0.05), inhibited (P<0.05) and unchanged when the cultured cells were treated with bFGF at the concentrations of 5, 10 and 15 ng/ml, respectively. The content and activity of FAK protein were markedly up-regulated in 5 ng/ml bFGF group and down-regulated in 15 ng/ml bFGF group, respectively. FAK mRNA expression came to the peak in 5 ng/ml bFGF group after 6 h culture and there was a significant difference compared with the control group. In various experimental groups there were no significant differences in the expression of integrin alpha3 compared with the control group according to the immunocytochemical staining. The results mentioned above suggest that different concentrations of bFGF have a dual-phase effect on the migration of cultured ECV-304 cells, which correlates positively with FAK content, activity and mRNA in cultured ECV-304 cell scratch wound model. The FAK plays an important role in the signal transduction pathway of cell migration induced by bFGF, while bFGF can regulate the content of FAK in ECV-304 cells at gene transcription level.
Cell Movement ; physiology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fibroblast Growth Factor 2 ; physiology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Integrin alpha3 ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; RNA, Messenger ; metabolism ; Umbilical Veins ; cytology

Erythropoietin-producing hepatoma (EPH) receptors are considered the largest family of receptor tyrosine kinases and play key roles in physiological and pathologic processes in development and disease. EPH receptors are often overexpressed in human malignancies and are associated with poor prognosis. However, the functions of EPH receptors in epithelial-mesenchymal transition (EMT) remain largely unknown. This review depicts the relationship between EPH receptors and the EMT marker E-cadherin as well as the crosstalk between EPH receptors and the signaling pathways involved EMT. Further discussion is focused on the clinical significance of EPH receptors as candidates for targeting in cancer therapeutics. Finally, we summarize how targeted inhibition of both EPH receptors and EMT-related signaling pathways represents a novel strategy for cancer treatment.
Antineoplastic Agents ; Cadherins ; Epithelial-Mesenchymal Transition ; Humans ; Neoplasms ; Receptor Protein-Tyrosine Kinases ; Receptors, Eph Family ; physiology ; Signal Transduction

AIMTo investigate if the phytoestrogen, genistein, affects essential functions of cryopreserved bovine spermatozoa.

METHODSThe effect of genistein upon motility was assessed by computer-assisted motion analysis. Hemizona assay was performed to detect the ability of spermatozoa binding to the zona pellucida. The inducibility of the acrosome reaction using progesterone and ZP3-6 peptide was analysed by fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA)/Hoechst 33258 double staining. Capacitation after incubation with genistein was assessed by the chlortetracycline (CTC) assay. Immunoblots showed the pattern of protein tyrosine phosphorylation of cryopreserved bovine spermatozoa.

RESULTSImmunodetection of tyrosine-phosphorylated proteins showed that genistein did not affect tyrosine phosphorylation in cryopreserved bovine spermatozoa. However, genistein significantly reduced the progesterone- and ZP3-6 peptide-mediated induction of the acrosome reaction and led to a dose-dependent inhibition of sperm-zona pellucida binding; while sperm motility and capacitation were not affected by this phytoestrogen, as indicated by computer-assisted sperm motion analysis and the CTC assay, respectively.

CONCLUSIONOur results suggest that in cryopreserved bovine spermatozoa, genistein affects a protein tyrosine phosphorylation-independent signal transduction pathway that is involved in sperm capacitation, the acrosome reaction and sperm-zona pellucida binding.

Acrosome Reaction ; drug effects ; physiology ; Animals ; Cattle ; Enzyme Inhibitors ; pharmacology ; Genistein ; pharmacology ; Male ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Sperm Capacitation ; drug effects ; physiology ; Zona Pellucida ; drug effects ; physiology

BACKGROUNDPulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein.

METHODSCultured HPASMCs stimulated by fibronectin (40 microg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expression of FAK, Jun NH2-terminal kinase (JNK), cyclin-dependent kinase 2 (CDK 2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining.

RESULTSWhen compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense-FAK ODNs group. The level of cell apoptosis in antisense-FAK group was higher than in the mismatch sense group and the latter was higher than sense-FAK group. In addition, the sense-FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense-FAK ODNs group was weakly stained.

CONCLUSIONSThe results suggest that FAK relates to the proliferation of HPASMCs. Antisense-FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis. It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase-3 inhibits HPASMCs apoptosis.

Apoptosis ; CDC2-CDC28 Kinases ; analysis ; Caspase 3 ; Caspases ; analysis ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase 2 ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Immunohistochemistry ; JNK Mitogen-Activated Protein Kinases ; analysis ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Protein-Tyrosine Kinases ; analysis ; physiology ; Pulmonary Artery ; cytology

OBJECTIVETo investigate the MEK and ERK expression and their relationship with clinicopathological parameters in human breast carcinoma, and the effect of preoperative chemotherapy on MEK and ERK protein expression.

METHODSSamples were obtained from 56 patients with breast carcinoma and 8 patients with benign tumors. Sixteen of the 56 patients received preoperative chemotherapy. Western blot and immunohistochemistry were used to measure the expression of MEK1, MEK2 and ERK1, ERK2 protein.

RESULTSMEK2 and ERK1, ERK2 protein levels were increased in breast carcinoma tissue compared with those in adjacent normal tissues (t = 7.244, 5.959, 3.735, P < 0.01) and benign tumors (t = 2.206, P < 0.05). The levels of MEK1 were decreased. The expression of MEK2 protein in ER negative patients was higher than that in ER positive ones. MEK2 protein levels were lower in patients who received preoperative chemotherapy than in those who did not.

CONCLUSIONOverexpression of MEK-ERK may play an important role in the development of human breast carcinoma. MEK and ERK protein expressions are inhibited by preoperative chemotherapy.

Adult ; Aged ; Blotting, Western ; Breast Neoplasms ; diagnosis ; enzymology ; metabolism ; Female ; Humans ; Immunohistochemistry ; MAP Kinase Kinase 1 ; MAP Kinase Kinase 2 ; MAP Kinase Signaling System ; physiology ; Middle Aged ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinase Kinases ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Prognosis ; Protein Kinases ; metabolism ; Protein-Serine-Threonine Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism

OBJECTIVE: To investigate the role of focal adhesion kinase (FAK) in extracellular signal-regulated kinase (ERK) signaling pathway mediated invadsion of trophoblasts. METHODS: We established a human extravillous cytotrophoblasts in vitro invasion model. Different concentrations of herbimycin A(FAK inhibitor)and PD98059 (ERK inhibitor) were given to observe the influence on the growth of trophoblast cells, FAK, ERK phosphorylation, and trophoblast invasion abilities. RESULTS: The expression of phosphorylated FAK in the extravillous cytotrophoblasts (EVCT) was inhibited by herbimycin A in a concentration-dependent manner and expression of phosphorylated ERK1/2 was also partially reduced. PD98059 had no effect on the expression of phosphorylated FAK. Herbimycin A and PD98059 suppressed the in vitro invasion of EVCT to various degrees. CONCLUSION ERK signaling pathway may be the common pathway for many invasive signals,and play a key role in the regulation of trophoblast invasion.
Benzoquinones ; pharmacology ; Cell Division ; physiology ; Cell Movement ; physiology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Flavonoids ; pharmacology ; Focal Adhesion Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Phosphorylation ; Rifabutin ; analogs & derivatives ; Signal Transduction ; physiology ; Trophoblasts ; cytology ; physiology

AIMTo investigate the effects of protein tyrosine kinase on the inflammation and airway remodeling in lung of guinea pigs with bronchial asthma.

METHODS30 adult male guinea pigs were randomly divided into 3 groups (n=3): control group (C group), asthmatic group(A group)and genistein group (B group). Asthmatic model was established by ovalbumin intraperitoneal injection and ovalbumin inhalation. The total cell and the proportion of inflammatory cell in bronchial alveolar lavage fluid(BALF), inflammatory cell infiltration and index of remodeling of bronchiole were measured, respectively. The expression of p-tyrosine in lung tissue was examined by immunohistochemistry.

RESULTSThe total cell and proportion of eosinophil in BALF of A group were significantly higher than that of C group (P < 0.01), but compared with A group, the total cell and proportion of eosinophil in BALF of B group were much lower (P < 0.01). The number of eosinophile and lymphocyte of bronchiole in A group were significantly higher than that of C group (P < 0.01), but compared with A group, the number of eosinophile and lymphocyte in bronchiole of B group were much lower (P < 0.01). Compared with A group, the remodeling of bronchiole of B group was significantly relieved (P <0.01), there was no difference between B and C group (P > 0.05). Immunohistochemistry indicated that in A group the p-tyrosine was more positively expressed at the bronchial smooth muscle, bronchial epithelium, smooth muscle of vessel and inflammatory cell, especially at smooth muscle of bronchi and vessel and inflammatory cell than that of C group (P <0.01), there was no difference between B group and C group (P > 0.05).

CONCLUSIONPTK played a key role in inflammation and bronchial remodeling in lung of guinea pigs with bronchial asthma. The Protein tyrosine kinase inhibitor genistein could prevent and inhibit the inflammation and bronchial remodeling in lung of guinea pigs with bronchial asthma.

Airway Remodeling ; physiology ; Animals ; Asthma ; physiopathology ; prevention & control ; Genistein ; pharmacology ; Guinea Pigs ; Inflammation ; prevention & control ; Male ; Ovalbumin ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; physiology ; Random Allocation