Source: Annual Congress 2003 - Molecular biology of chronic lung disease and lung cancer
Disease area: Thoracic oncology

Abstract

PAI-1 is one of the key regulators of tumor growth, metastasis and cancer angiogenesis. However, wPAI-1 rapidly looses its anti-urokinase (uPA) activity with half-life time (T_1/2) of 30‘.Based on known structure of active PAI-1 we have identified preferable localization of disulfide bridges that should prevent PAI-1 molecule inactivation.Desired mutations were introduced by PCR method.Proteins were expressed in E.coli transfected with the pTYB12 plasmid containing PAI-1/intein gene.PAI-1s were purified using intein affinity binding column.Functional activity of cysPAI-1s was proven by complex formation assay.Their T_1/2 was significantly higher in comparison to wPAI-1 with strongest effect observed for mutation in A3,A5 region (T_1/2of~7000h).Specific inhibitory activity to uPA and inhibitory constant (by Lineweaver-Burke analysis) were very similar for wPAI-1 and two representative cysPAI-1s with moderate (190h) and strong (~7000h) increase of T_1/2.Both cysPAI-1s revealed significant antiangiogenic dose-dependend activity suppressing in vitro HUVEC and HMVEC-L sprouts formation.That effect was stronger for the mutant with T_1/2~7000h.Development of stable highly selective uPA inhibitors might be vital for the researches on a new class of anticancer agents.Since HMVEC-Ls are the first-line lung cells interacting with metastatic cancer cells and are known for the high production of uPA, cysPAI-1 might be especially effective in the treatment of lung tumors.

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J. Chorostowska-Wynimko, R. Swiercz, E. Skrzypczak-Jankun, A. Wojtowicz, S. Selman, J. Jankun (Warsaw, Poland; Smithville, Toledo, United States Of America). Plasminogen activator inhibitor-1 (PAI-1) mutations of extended half-life time inhibit angiogenic properties of human lung microvascular endothelial cells (HMVEC-L). Eur Respir J 2003; 22: Suppl. 45, 1507

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