Abstract
Introduction: The restriction of prolyl-protein cis/trans isomerase 1 (Pin1) activity has been shown to prevent the release of tissue factor (TF) leading to the accumulation of the latter protein within the cell. This study tested the ability of novel small molecules to inhibit Pin1, suppress TF activity and release, and induce cellular apoptosis. Methods: Four compounds were designed and synthesised based on modification of 5-(p-methoxyphenyl)-2-methylfuran-3-carbonyl amide and the outcome on MDA-MB-231 and primary cells examined. These compounds contained 3-(2-naphthyl)-D-alanine (4a), D-tryptophan (4b), D-phenylalanine (4c), and D-tyrosine (4d) at the amino-termini. Results: Treatment of cells with compound 4b and 4d reduced the cell-surface TF activity after 60 min on MDA-MB-231 cells. Incubation with compound 4d also reduced TF antigen on the cell surface and its incorporation into microvesicles, while compounds 4a and 4b significantly increased TF release. None of the four compounds significantly altered the total amount of TF antigen or TF mRNA expression. Compound 4b and 4d also suppressed the binding of Pin1 to TF-cytoplasmic domain peptide. However, compound 4d reduced while compound 4b increased the Pin1 isomerase activity. Finally, treatment with compound 4b and 4d reduced the cell numbers, increased nuclear localisation of p53, Bax protein and bax mRNA expression and induced cellular apoptosis in MDA-MB-231 but not primary endothelial cells. Conclusions: In conclusion, we have identified small molecules to regulate the function of TF within cells. Two of these compounds may prove to be beneficial in moderating TF function specifically and restrain TF-mediated tumour growth without detrimental outcomes on normal vascular cells.
Highlights
Regulation of tissue factor (TF) activity and its release as microvesicles is imperative to ensure adequate coagulation during injury, without endangering the precipitation of thrombosis
The inability of cells to dispose of excess TF efficiently appears to contribute to the induction of cell apoptosis, mediated via p53 nuclear localisation and the expression of Bax protein [24, 27, 28]
It has been established that prolyl-protein cis/trans isomerase (Pin1) is capable of influencing p53 function directly [13, 29,30,31,32], there appears to be an independent regulation mediated through TF
Summary
Regulation of tissue factor (TF) activity and its release as microvesicles is imperative to ensure adequate coagulation during injury, without endangering the precipitation of thrombosis. Pin is a regulator of post-phosphorylation processes and binds to the phosphoserine-proline (termed an MPM-2) motif [6,7,8,9,10,11,12,13,14,15]. The rotation of the peptide bond between the phosphoserine and the proline may be facilitated by enzymes including Pin. The action of Pin on the cytoplasmic domain of TF prevents the de-phosphorylation of serine 253 within the cytoplasmic domain of TF. This extends the release of TF within microvesicles by preventing TF ubiquitination [4, 20]. It has been established that Pin is capable of influencing p53 function directly [13, 29,30,31,32], there appears to be an independent regulation mediated through TF
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