ABSTRACT
<p><b>BACKGROUND</b>Tumstatin is a novel endogenous angiogenesis inhibitor which is widely studied using purified protein. The current study evaluates the antiangiogenic effects of tumstatin-overexpression plasmid in vitro, reveals the mechanism underlying the vascular endothelial cell growth inhibition and searches for a novel method administering tumstatin persistently.</p><p><b>METHODS</b>The eukaryotic expression plasmid pcDNA-tumstatin encoding tumstatin gene was constructed and transfected to human umbilical vein endothelial cell ECV304 and human renal carcinoma cell ACHN. Expression of tumstatin in the two cell lines was determined by RT-PCR and Western blotting. Vascular endothelial cell proliferation was assessed by CCK-8 assay and cell cycle was analyzed by flow cytometry. To investigate the mechanism by which pcDNA-tumstatin inhibited vascular endothelial cell proliferation in vitro, cyclin D1 protein was detected by Western blotting.</p><p><b>RESULTS</b>DNA sequence confirmed that pcDNA-tumstatin was successfully constructed. RT-PCR and Western blotting indicated that tumstatin could express in the two cell lines effectively. After tumstatin gene transfer, ECV304 cell growth was significantly inhibited and the cell cycle was arrested in G1 phase. And Western blotting showed that pcDNA-tumstatin decreased the level of cyclin D1 protein.</p><p><b>CONCLUSIONS</b>Overexpression of tumstatin mediated by pcDNA 3.1 (+) specially inhibited vascular endothelial cells by arresting vascular endothelial cell in G1 phase resulting from downregulation of cyclin D1 and administration of tumstatin using a gene therapy might be a novel strategy for cancer therapy.</p>
Subject(s)
Humans , Autoantigens , Genetics , Metabolism , Blotting, Western , Cell Cycle , Genetics , Physiology , Cell Line , Cell Line, Tumor , Cell Proliferation , Collagen Type IV , Genetics , Metabolism , Endothelial Cells , Cell Biology , Metabolism , Flow Cytometry , Plasmids , Genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To study the inhibitory effects of antisense bicistronic recombinant adenovirus vector of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (Ad-ODC-AdoMetDCas) on polyamine biosynthesis,proliferation and invasion of lung cancer cells.</p><p><b>METHODS</b>Adenovirus-mediated gene transduction efficiency was assessed with counting GFP-positive cells using trypan blue. Western Blot and HPLC were used to detect ODC and S-AdoMetDC expression and polyamine content in A-549 cells respectively. Viable cell counting and cell cycle analysis were adopted to evaluate cell growth and cell cycle distribution, and A-549 cell invasion in vitro was detected with Matrigel invasion assay.</p><p><b>RESULTS</b>Approximate 75% of A-549 cells were infected with Ad-ODC-AdoMetDCas when multiplicity of infection reached 50. Our study demonstrated that Ad-ODC-AdoMetDCas vector-mediated gene transfer inhibited tumor cell growth through the blockade of polyamine synthesis pathway. The tumor cells were arrested at cell cycle G1 phase after gene transfer. Gene transferred tumor cells were shown to possess markedly decreased invasiveness.</p><p><b>CONCLUSION</b>Ad-ODC-AdoMetDCas has significant inhibitory effects on lung cancer cell proliferation and invasion and bears therapeutic potential for the treatment of lung cancer.</p>
Subject(s)
Humans , Adenosylmethionine Decarboxylase , Genetics , Metabolism , Adenoviridae , Genetics , Blotting, Western , Cell Cycle , Cell Line, Tumor , Cell Movement , Cell Proliferation , Chromatography, High Pressure Liquid , Genetic Vectors , Green Fluorescent Proteins , Genetics , Metabolism , Lung Neoplasms , Genetics , Metabolism , Pathology , Ornithine Decarboxylase , Genetics , Metabolism , Polyamines , Metabolism , RNA, Antisense , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate relationship between the expression of heparanase (HPSE) and vascular endothelial growth factor-C (VEGF-C) and tumorigenesis, progression in human lung cancer.</p><p><b>METHODS</b>The expression of HPSE and VEGF-C protein in 65 cases of lung cancer, adjacent tissues of cancer and normal tissues was tested by immunohistochemical SABC method and analysed by clinico-pathological characteristics and prognosis of lung cancer.</p><p><b>RESULTS</b>The rate of expression of HPSE and VEGF-C protein in tumor tissues (51% and 57%) was significantly higher than that in adjacent tissues of cancer (9% and 12%) and normal tissues (5% and 6%) (chi2 = 34.6, 38.8, 26.7, 28.6; P < 0.01); It was shown that HPSE and VEGF-C protein expression did significantly not correlate with the type (chi2 = 0.39, 0.41, P > 0.05) and grade of the tumor (chi2 = 0.45, 0.04, P > 0.05); but it correlated with the clinical stage (chi2 = 26.6, 20.1; P < 0.01) and survival time of the patients (chi2 = 21.5, 22.2; P < 0.01).</p><p><b>CONCLUSIONS</b>The results suggest that there be overexpression of HPSE and VEGF-C protein in lung cancer tissues, and which perhaps participate in regulation of tumorigenesis, progression in lung cancer. The expressions of HPSE and VEGF-C protein are used as an useful marker of the biological behavior of lung cancer and as an independent prognosis factor for the patients with lung cancer.</p>