Khezerdost S, Bahadori F, Shafaat M, Yahyazadeh H, Yahyazadeh N, Amini E,
Volume 66, Issue 10 (1-2009)
Background: Tumor cells need food and oxygen supply for growth and division. Therefore one of the most promising areas of cancer therapy focuses on using agents that inhibit tumor angiogenesis. Inhibition of angiogenesis prevents cell growth, division and metastasis. Previous studies showed that plasminogen related Protein-B has an anti-tumor activity in mice. This protein has a high level of homology with preactivation Peptide (PAP) of human plasminogen. According to this high homology, antiangiogeneic activity of PAP was investigated in an in vitro angiogenesis model.
Methods: PAP encoding region of human plasminogen gene was isolated by Polymerase Chain Reaction and cloned in pGEX-2T vector. This plasmid was expressed in Escherichia coli as a fusion protein (GST-PAP). GST-PAP was expressed as inclusion body and purified by affinity chromatography on GSH-sepharose resin after refolding. antiangiogenic effects of purified protein were surveyed with Matrigel assay.
Results: The GST-PAP was expressed and purified and its accuracy was confirmed by SDS-PAGE analysis and immunoblotting. Microscopic studies showed that GST-PAP inhibited angiogenesis in Matrigel system which is shown by shrinking the length of capillary like structures and a decrease in the number of tubule. While applying concentarations of 25μg/ml of GST-PAP and concentrations above that, antiangiogenic activity of GST-PAP was significant comparing to the controls.
Conclusion: Finding shows that GST-PAP can inhibit network formation in Matrigel system. This findings support the theory that PAP is a potent angiogenesis inhibitor.
