Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yang
Ya-pei
YP
Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan, Shandong 250012, China.
Xu
Chun-xiao
CX
Hou
Guo-sheng
GS
Xin
Jia-xuan
JX
Wang
Wei
W
Liu
Xian-xi
XX
D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism*; D015153:Blotting, Western; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D024141:Collagen Type IV / Q000235:genetics* / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001324:Autoantigens; D024141:Collagen Type IV; C057112:type IV collagen alpha3 chain