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

OBJECTIVETo investigate the effect and mechanisms of tumor suppressor gene PTEN on the induction of anoikis of hepatocellular carcinoma SMMC-7721 cells.

METHODSSMMC-7721 cells were transfected with GFP plasmids containing wild-type PTEN or phosphatase inactivating mutant PTEN (C124A-PTEN) in vitro; The PTEN expression and the phosphorylation levels of focal adhesion kinase (FAK) and protein kinase B (PKB/Akt) were detected by Western blotting; Flow cytometry assay and laser scanning confocal microscopy were used to analyze apoptosis in adherent and non-adherent cells.

RESULTSCompared with the control, PTEN expression in the cells transfected with wild-type PTEN increased to 248%, while the phosphorylation level of FAK and Akt decreased 65.2% and 89.1%, respectively; and the anoikis percentage increased from 9.5% to 31.3%. In the cells transfected with C124A-PTEN, neither the phosphorylation of FAK and Akt nor the anoikis percentage had obviously changed, although the PTEN expression enhanced dramatically in comparison with the control.

CONCLUSIONThrough its phosphatase activity, tumor suppressor gene PTEN can suppress the phosphorylation of FAK and Akt, and induce anoikis in hepatocellular carcinoma cells.

Anoikis ; physiology ; Carcinoma, Hepatocellular ; pathology ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Liver Neoplasms ; pathology ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; Phosphoric Monoester Hydrolases ; metabolism ; Phosphorylation ; Tumor Cells, Cultured

OBJECTIVETo study the role of focal adhesion kinase (FAK) in the pathogenesis of human hypertrophic scar.

METHODSHuman hypertrophic scar fibroblasts (HSFB) were isolated from human hypertrophic scar and cultured in vitro. The cells were then divided into 3 groups as AT group (phosphorothioate FAK ASODN was transfected into the HSFB by liposome), LPC group (liposome only), and LC group (control group, without liposome or ASODN). The FAKmRNA index of HSFB was assessed by polymerase chain reaction method (FQ-PCR). The collagen synthesis of HSFB was assessed by 3H-proline incorporation method.

RESULTSThe FAK mRNA index of HSFB in AT group 48 hours after transfection was significantly lower than that in LPC and LC groups (0.043 +/- 0.030, 0.124 +/- 0.070, 0.127 +/- 0.0195, P < 0.05). The 3H-proline incorporation rate in AT group was lower than that in LPC and LC groups (257.0 +/- 15.14, 962.2 +/- 300.5, 930.8 +/- 28.97, P < 0.01).

CONCLUSIONThe expression of FAK gene and collagen synthesis of the cultured HSFB could be inhibited by FAK ASODN, indicating that FAK played a role in the development of excessive fibrosis of human hypertrophic scar.

Cells, Cultured ; Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Collagen ; biosynthesis ; Fibroblasts ; drug effects ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; genetics ; metabolism ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; Transfection

AIMTo study the effects of FAK-ERK1/2 signaling pathway and FAK antisense oligodeoxynucleotides (ODNs) on vascular smooth muscle cell (SMC) migration and adhesion stimulated by fibronectin (FN).

METHODSMigration and adhesion of cultured SMCs were stimulated by different concentrations of FN, FAK, ERK1/2. And their phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense ODNs were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation, SMCs migration and adhesion were also measured by modifing Boyden Chamber and morphological enumeration, respectively.

RESULTSFAK were expressed when SMCs adhesion and migration were successfully simulated by FN (5, 10, 20, 40, 60 micrograms.mL-1), high contents of FAK and ERK1/2 phosphorylation were detected by 20 micrograms.mL-1 FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid. FAK and ERK1/2 phosphorylation were inhibited magnificently after FAK antisense ODNs transfection. Cell migration stimulated by FN 10, 20, 40 and 60 micrograms.mL-1 were reduced by 23.26%, 21.63%, 19.31% and 17.88% respectively (P < 0.05). SMCs adhesive spreading in 5-60 micrograms.mL-1 FN groups were reduced by 17.89%-27.67% (P < 0.05).

CONCLUSIONFAK-ERK1/2 mediated signal transduction play important roles in SMCs migration and adhesion stimulated by extracellular matrix. The process can be inhibited by FAK antisense ODNs effectively.

Animals ; Aorta ; cytology ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Fibronectins ; pharmacology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; biosynthesis ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Transfection

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases; Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.
Bronchi/*cytology ; Bronchi/metabolism ; Cell Cycle/drug effects ; Cell Proliferation ; Cells, Cultured ; Enzyme Activation ; Epithelial Cells/*cytology ; Epithelial Cells/enzymology ; Focal Adhesion Protein-Tyrosine Kinases/biosynthesis ; Focal Adhesion Protein-Tyrosine Kinases/*metabolism ; Phosphorylation ; Tobacco/adverse effects ; Tobacco Smoke Pollution/*adverse effects

Hepatic stellate cell (HSC) plays a pivotal role in liver fibrosis and isconsidered as one of the therapeutic targets for the treatment of hepatic fibrosis. Focal adhesion kinase (FAK) has been shown to play an important role in the HSC activation. The aim of the study is to explore the role of FAK in the proliferation and activation in culture-activated rat HSCs by using a specific antisense oligonucleotides targeting FAK (FAK-ASON). Rat HSCs were prepared from SD rats by in situ perfusion of pronase and collagenase and single-step density Nycodenze gradient. Culture-activated HSCs were transfected with the FAK-ASON (1 microM) by lipofectamine 2000 for 24, 48 or 72 hours. The proliferation of HSC was detected by MTT assay. The expression of the marker of HSC activation, alpha-smooth muscle actin (alpha-SMA), was assessed by reverse transcription- polymerase chain reaction (RT-PCR) and Western blot. The inhibition rates for HSC proliferation 24, 48 and 72 hours after transfection were 65.5% +/- 5.8%, 46.8% +/- 4.3% and 35.7% +/- 5.2% respectively. Transfection of FAK-ASON could significantly inhibit the proliferation of HSC. Meanwhile, treatment with the FAK-ASON could markedly decrease the mRNA and protein expression of alpha-SMA in rat HSC. The specific FAK-ASON may have an inhibitory effect on the proliferation and activation in rat HSC.
Actins ; biosynthesis ; genetics ; Animals ; Cell Proliferation ; Cells, Cultured ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Hepatocytes ; cytology ; enzymology ; Male ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection