ABSTRACT
BACKGROUND & OBJECTIVE: Loss of activity of death- associated protein kinase 1 (DAPK1) may be an independent factor affecting survival of non-small cell lung cancer patients. DAPK1 over-expression can induce cell apoptosis and inhibit tumor cell metastasis. However, the mechanism of DAPK1 inhibiting metastasis was unclear yet. This study was designed to investigate the mechanism of DAPK1 affecting PGCl3 cells' growth and metastasis. METHODS: Open read frame (ORF) of DAPK1 gene was recombined into eukaryotic express vector pcDNA3.1. The PGCl3 cell line, a high metastasis lung tumor cell line, was transfected with pcDNA3.1-DAPK1 by lipofectamine2000. The growth curve of PGCl3 cells, colony formation assays, invasive, migration and adhesion ability were evaluated. Gelatinase secretion of PGCl3 cells treated with DAPK1 and expression of p53 and bcl-2 gene in PGCl3 cells were determined. RESULTS: PGCl3 cells of pcDNA3.1-DAPK1-transfected group grew slower than blank group and pcDNA3.1-transfected group. The numbers of colony formation of pcDNA3.1-DAPK1-transfected group reduced in comparison with blank group and pcDNA3.1-transfected group (P< 0.05). Invasive ability of pcDNA3.1-DAPK1-transfected group was 68.5% to blank group, and pcDNA3.1-transfected group was 88% to blank group. Migration ability of pcDNA3.1-DAPK1-transfected group was 87.3% to blank group and pcDNA3.1-transfected group was 95.7% to blank group. Adhesion ability of pcDNA3.1-DAPK1-transfected group was 62.7% to blank group, and pcDNA3.1-transfected group was 91.2% to blank group. Changes in PGCl3 cells secreting gelatinase have not been observed in different groups. Expression of p53 gene was upregulated in pcDNA3.1-DAPK1-transfected group, while expression of bcl-2 gene was downregulated. CONCLUSION: DAPK1 gene over-expression could suppress PGCl3 cells malignant phenotype, inhibit PGCl3 cells growth, invasive, migration and adhesion ability, upregulate p53 gene and downregulate bcl-2 gene. All of these changes may be the mechanism of inhibition of non-small cell lung cancer metastasis induced by DAPK1.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , Protein Serine-Threonine Kinases , Transfection , Apoptosis Regulatory Proteins , Calcium-Calmodulin-Dependent Protein Kinases , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Death-Associated Protein Kinases , Gene Expression Regulation, Neoplastic , Genes, bcl-2 , Genes, p53 , Genetic Vectors , Humans , Lung Neoplasms/metabolism , Neoplasm Invasiveness , Neoplasm Metastasis/physiopathology , Open Reading Frames , Protein Serine-Threonine Kinases/biosynthesis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Recombination, GeneticABSTRACT
OBJECTIVE: To obtain purified deletion mutant of plasminogen kringle 5 (K5) using gene mutation and genetic recombination methods and assess its anti-angiogenic activity in vitro. METHODS: A deletion mutant of K5 was obtained by deleting 15 amino acids from K5 while retaining all the 3 disulfide bonds. This K5 mutant (Mut1) was expressed in E. coli and affinity purified. The inhibition effect of K5 Mut1 on primary retinal capillary endothelial cells and pericytes from the same origin was assessed by MTT assay. RESULTS: The K5 Mut1 inhibited the proliferation of primary retinal capillary endothelial cells in a concentration-dependent manner, with an apparent half-inhibition concentration (EC(50)) of approximately 35 nmol/L, which was 2-fold more potent than intact K5. In the same concentration range, this peptide did not inhibit pericytes from the same origin, suggesting an endothelial cell-specific inhibition. CONCLUSION: This K5 deletion mutant is a more potent angiogenic inhibitor than K5 and may have therapeutic potential in the treatment of such disorders with abnormal neovascularization as diabetic retinopathy, age-related macular degeneration and solid tumor.
