OBJECTIVETo investigate the role of caveolin-1 down-regulation in human hepatocyte proliferation in vitro.

METHODSThe expression vector psiRNA-CAV1 was constructed and transfected into Chang liver cells (CHL). The caveolin-1 down-regulated cell clones were selected by the antibiotic zeocin. The proliferation of the cell strain CAV7 was examined by MTT, in which untransfected CHL and HepG2 cells were set as controls. Expression of caveolin-1, Akt, Erk1/2, p-Akt and p-Erk1/2 in the transfected and control cells was detected by Western blot.

RESULTSAfter caveolin-1 expression was down-regulated by RNAi, CHL increased faster at first (24 h and 72 h, P<0.05; 96 h, P<0.01), but slower later. P-Akt and p-Erk1/2 expressions were down-regulated, indicating that the growth and proliferation related Akt and Erk1/2 pathways were inhibited after caveolin-1 down-regulation.

CONCLUSIONCaveolin-1 may play an important role in hepatocyte proliferation.

Caveolin 1 ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Down-Regulation ; Hepatocytes ; cytology ; Humans ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA Interference ; RNA, Small Interfering ; Signal Transduction ; Transfection

OBJECTIVETo investigate the effects of caveolin-1 on the biologic behavior of laryngeal squamous cell carcinoma HEp2 cell line in vitro.

METHODSEukaryotic expression vector of human caveolin-1 gene was constructed and transfected into HEp2 cells by Lipofectamine. The clones stably overexpressing caveolin-1 were identified by real-time PCR and Western blotting. Cell proliferation viability was tested by MTT assay. Anchorage-independent growth was determined by assaying colony formation in soft agar. Flow cytometry was used to assess the cell cycle and apoptosis. The relative phosphorylation level of EGFR and ERK1/2 were detected by Western blotting. Localization of caveolin-1 and EGFR were studied by laser confocal laser scanning microscopy.

RESULTSThe expression vector of caveolin-1 was constructed and three clones stably overexpressing caveolin-1 were obtained. Comparing with the parental HEp2 cells, the transfected cells exhibited a slower growth rate and formed fewer colonies in soft agar. The results of FACS analysis revealed that overexpression of caveolin-1 resulted in the cell cycle arrest at G0/G1 phase and increased the apoptotic cell fraction. EGFR was found to colocalize with caveolin-1 in transfected cells by confocal laser scanning microscopy and Western blotting results showed that overexpression of caveolin-1 reduced the phosphorylation of EGFR and Erkl/2.

CONCLUSIONOverexpression of caveolin-1 suppresses the growth of HEp2 cells and induces apoptosis and inhibition of EGFR-MAPK signaling pathway may be involved in its mechanism.

Apoptosis ; Blotting, Western ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Caveolin 1 ; genetics ; metabolism ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Flow Cytometry ; Genetic Vectors ; chemistry ; genetics ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Lipids ; chemistry ; Microscopy, Confocal ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; Polymerase Chain Reaction ; methods ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; physiology ; Transfection ; methods

The purpose of this study was to demonstrate the cellular localization of cyclooxygenase-2 (COX-2) and caveolin-3 (Cav-3) in primarily cultured rat chondrocytes. In normal rat chondrocytes, we observed relatively high levels of Cav-3 and a very low level of COX-2 mRNA and protein. Upon treating the chondrocytes with 5 microM of CdCl2 (Cd) for 6 hr, the expressions of COX-2 mRNA and protein were increased with the decreased Cav-3 mRNA and protein expressions. The detergent insoluble caveolae-rich membranous fractions that were isolated from the rat chondrocytes and treated with Cd contained the both proteins of both COX-2 and Cav-3 in a same fraction. The immuno-precipitation experiments showed complex formation between the COX-2 and Cav-3 in the rat chondrocytes. Purified COX-2 with glutathione S-transferase-fused COX-2 also showed complex formation with Cav-3. Confocal and electron microscopy also demonstrated the co-localization of COX-2 and Cav-3 in the plasma membrane. The results from our current study show that COX-2 and Cav-3 are co-localized in the caveolae of the plasma membrane, and they form a protein-protein complex. The co-localization of COX-2 with Cav-3 in the caveolae suggests that the caveolins might play an important role for regulating the function of COX-2.
Animals ; Animals, Newborn ; Blotting, Western ; Cadmium Chloride/pharmacology ; Caveolae/drug effects/metabolism/ultrastructure ; Caveolin 3/*genetics/metabolism ; Cell Membrane/drug effects/metabolism ; Cells, Cultured ; Chondrocytes/cytology/drug effects/*metabolism ; Cyclooxygenase 2/*genetics/metabolism ; Gene Expression ; Immunoprecipitation ; Microscopy, Confocal ; Microscopy, Electron ; RNA, Messenger/genetics/metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the relationship between expression of caveolin-1 (Cav-1) and pERK1/2 and prognosis in non-small cell lung cancer (NSCLC).

METHODSCav-1 and pERK1/2 protein expression was assessed by immunohistochemistry in samples obtained from 160 patients with NSCLC and 20 patients with normal lung tissue.

RESULTSNormal bronchial and alveolar epithelial cells were positive for Cav-1 (membranous and cytoplasmic staining patterns). The expression rate of Cav-1 in NSCLC was 65.6% (105/160), which was significantly lower than that in normal lung tissue (P = 0.002). The Cav-1-positive rates in well to moderately differentiated tumors and poorly differentiated tumors were 56.8% (46/81) and 75.7% (53/70), respectively (P = 0.015). The expression of Cav-1 was much higher in patients with lymph node metastasis (77.8%, compared with 55.7% in lymph node-negative group, P = 0.003). The expression was also higher in stage III to IV than in stage I to II disease (75.4%, compared with 58.2%, P = 0.024). The overall survival of patients with Cav-1-positive tumors (71.4%, 37.1% and 17.1% 1-, 3- and 5-year survival, respectively) was lower than those with Cav-1-negative tumors (89.1%, 69.1% and 43.6% 1-, 3- and 5-year survival, respectively, P = 0.000). On the other hand, normal bronchial and alveolar epithelial cells were negative for pERK1/2. The expression rate of pERK1/2 in NSCLC was 61.3%, which was significantly higher than that in normal lung tissues (P = 0.000). The pERK1/2-positive rates in well to moderately differentiated tumors and poorly differentiated tumors was 53.1% and 71.4%, respectively (P = 0.021). The expression of pERK1/2 was much higher in patients with lymph node metastasis (80.6%, compared with 45.5% in lymph node-negative group, P = 0.000). The expression of pERK1/2 was also higher in stage III to IV than in stage I to II disease (76.8%, compared with 49.5%, P = 0.426). The overall survival of patients with pERK1/2-positive tumors (74.5%, 42.9% and 19.4% 1-, 3- and 5-year survival, respectively) was lower than those with pERK1/2-negative tumors (82.3%, 56.5% and 37.1% 1-, 3- and 5-year survival, respectively, P = 0.002). Cav-1 and pERK1/2 expression showed negative correlation (P = 0.000).

CONCLUSIONSCav-1 expression is lower in NSCLC than in normal lung tissue, whereas pERK1/2 expression is higher in NSCLC. Positive expression of Cav-1 and overexpression of pERK1/2 correlates with tumorigenesis and tumor progression of NSCLC. Cav-1 and pERK1/2 may serve as potential markers for predicting prognosis in NSCLC.

Carcinoma, Non-Small-Cell Lung ; diagnosis ; metabolism ; Caveolin 1 ; genetics ; metabolism ; Cytoplasm ; Humans ; Immunohistochemistry ; methods ; Lung Neoplasms ; diagnosis ; metabolism ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; diagnosis ; pathology ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Neoplasm Staging ; classification ; Prognosis

Dysferlin is a plasma membrane protein of skeletal muscle whose deficiency causes Miyoshi myopathy, limb girdle muscular dystrophy 2B and distal anterior compartment myopathy. Recent studies have reported that dysferlin is implicated in membrane repair mechanism and coimmunoprecipitates with caveolin 3 in human skeletal muscle. Caveolin 3 is a principal structural protein of caveolae membrane domains in striated muscle cells and cardiac myocytes. Mutations of caveolin 3 gene (CAV3) cause different diseases and where caveolin 3 expression is defective, dysferlin localization is abnormal. We describe the alteration of dysferlin expression and localization in skeletal muscle from a patient with raised serum creatine kinase (hyperCKaemia), whose reduction of caveolin 3 is caused by a CAV3 P28L mutation. Moreover, we performed a study on dysferlin interaction with caveolin 3 in C2C12 cells. We show the association of dysferlin to cellular membrane of C2C12 myotubes and the low affinity link between dysferlin and caveolin 3 by immunoprecipitation techniques. We also reproduced caveolinopathy conditions in C2C12 cells by a selective p38 MAP kinase inhibition with SB203580, which blocks the expression of caveolin 3. In this model, myoblasts do not fuse into myotubes and we found that dysferlin expression is reduced. These results underline the importance of dysferlin-caveolin 3 relationship for skeletal muscle integrity and propose a cellular model to clarify the dysferlin alteration mechanisms in caveolinopathies.
Animals ; Biopsy ; Caveolin 3 ; Caveolins/*genetics/metabolism ; Cell Line ; Creatine Kinase/*blood ; Enzyme Inhibitors/*pharmacology ; Humans ; Imidazoles/pharmacology ; Insulin/pharmacology ; Membrane Proteins/*metabolism ; Mice ; Mitogen-Activated Protein Kinases/*antagonists & inhibitors/metabolism ; Muscle Proteins/*metabolism ; Muscle, Skeletal/cytology/metabolism ; Mutation/*genetics ; Protein Binding ; Pyridines/pharmacology ; p38 Mitogen-Activated Protein Kinases