ABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of SOCS3 and Pyk2 and their correlations in non-small cell lung cancer (NSCLC).</p><p><b>METHODS</b>The expression of SOCS3 and Pyk2 was detected in 100 cases of NSCLC, human bronchial epithelial cells (HBE) and 6 lung cancer cell lines by immunohistochemistry and immunofluorescence staining. The methylation status of SOCS3 was investigated in A549 cells by methylation-specific PCR. A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine (5-aza) or transfected with three SOCS3 mutants with various functional domains deleted. Besides, the cells were pretreated with a proteasome inhibitor β-lactacystin where indicated. The effects of SOCS3 on Pyk2 expression, Pyk2 Tyr 402 and ERK1/2 phosphorylations were assessed by Western blot. RT-PCR was used to estimate Pyk2 mRNA levels. Transwell experiments were performed to evaluate cell migration.</p><p><b>RESULTS</b>SOCS3 (43.0%, 43/100) and Pyk2 (65.0%, 65/100) were expressed in NSCLC. A significant negative correlation was found between SOCS3 and Pyk2 in both NSCLC tissues and cell lines. SOCS3 was aberrantly methylated and 5-aza restored SOCS3 expression. Transfection studies indicated that exogenous SOCS3 interacted with Pyk2, and both Src homology 2 (SH2) and kinase inhibitory region (KIR) domains contributed to Pyk2 binding. Furthermore, SOCS3 was found to inhibit Pyk2-associated ERK1/2 activity in A549 cells. SOCS3 possibly promoted degradation of Pyk2 in a SOCS-box-dependent manner and interfered with cell migration.</p><p><b>CONCLUSIONS</b>The data indicates that SOCS3 definitely plays roles in regulating Pyk2 signaling, and cell motility. Decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells.</p>
Subject(s)
Humans , Adenocarcinoma , Genetics , Metabolism , Pathology , Carcinoma, Non-Small-Cell Lung , Genetics , Metabolism , Pathology , Carcinoma, Squamous Cell , Genetics , Metabolism , Pathology , Cell Line, Tumor , Cell Movement , DNA Methylation , Focal Adhesion Kinase 2 , Metabolism , Lung Neoplasms , Genetics , Metabolism , Pathology , Lymphatic Metastasis , Mutation , Phosphorylation , Signal Transduction , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Genetics , MetabolismABSTRACT
This study was performed in China Medical University Shenyang, China from September 2007-February 2008. The design of the study was to modify DCs with GPC3 and to be used to activate human T cells and elicit a cell-mediated immune response against HepG2 in vitro. The GPC3 gene expression was identified by western blot and immunocytochemistry. The proliferation of responder cells and cytotoxicity against HepG2 were examined by water-soluble tetrazolium salt -1 and lactate dehydrogenase assay respectively. The interferon-y [IFN-gamma] secreted was detected by ELISA assay. Both Western blot and immunocytochemical analysis assured the validity of GPC3 transfection. Glypican3 modified DCs were potent in inducing responder cells proliferation and IFN-gamma production. The cytotoxicity in the group of GPC3 transfected DCs were [38.90 +/- 0.95%] at the ratio of effector cells/target cells E/T:100:1, 30.83 +/- 1.24% at the ratio of E/T:50:1, and 23.84 +/- 0.65% at the ratio of E/T:10:1, respectively [which is significant compared with other groups, p<0.001]. And the GPC3 modified DCs showed ability to induce high specific cytotoxicity against HepG2 in vitro. The effector cells stimulated with DCs that were transfected with pEF-hGPC3 plasmid could effectively lyse GPC3 expressing HepG2 cells, which suggested that those genetically engineered DCs have the potential to serve as novel vaccine for HCC