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Published on: December 3, 2015
Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro
Ya-pei Yang1, Chun-xiao Xu, Guo-sheng Hou
1Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan, Shandong 250012, China.
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.
Insights
Gene therapy using tumstatin overexpression plasmid effectively inhibited vascular endothelial cell growth by arresting the cell cycle. This novel approach, targeting cyclin D1, shows promise for cancer treatment.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Tumstatin is a known endogenous angiogenesis inhibitor.
- Previous studies focused on purified tumstatin protein.
- This study investigates tumstatin-overexpression plasmid for antiangiogenic effects.
Purpose of the Study:
- Evaluate in vitro antiangiogenic effects of tumstatin-overexpression plasmid.
- Elucidate the mechanism of vascular endothelial cell growth inhibition.
- Explore persistent tumstatin administration via gene therapy.
Main Methods:
- Constructed and transfected pcDNA-tumstatin plasmid into ECV304 and ACHN cells.
- Assessed tumstatin expression using RT-PCR and Western blotting.
- Analyzed cell proliferation (CCK-8), cell cycle (flow cytometry), and cyclin D1 levels (Western blotting).
Main Results:
- Successfully constructed and expressed tumstatin via pcDNA-tumstatin.
- Significantly inhibited ECV304 cell growth and arrested cell cycle at G1 phase.
- Demonstrated tumstatin overexpression downregulated cyclin D1 protein levels.
Conclusions:
- pcDNA-tumstatin effectively inhibits vascular endothelial cells by arresting the G1 phase.
- Downregulation of cyclin D1 is a key mechanism in tumstatin-mediated growth inhibition.
- Gene therapy with tumstatin presents a novel strategy for cancer treatment.
