Tissue factor activity of SW-480 human colon adenocarcinoma cells is modulated by thrombin and protein kinase C activation

Abstract

Expression of tissue factor (TF), a cellular initiator of the extrinsic coagulation cascade, is a feature of many malignant tumours and is intimately involved in the process of metastasis. SW-480 human colon adenocarcinoma cells responded to thrombin (1 U ml(-1)) or phorbol 12-myristate 13-acetate (PMA, 0.1 microM) with a 6.0-fold and a 7.7-fold increase in their procoagulant activity (PCA), respectively, after 4-6 h incubation in serum-free medium. The thrombin-enhanced PCA was significantly inhibited by complexing of thrombin with hirudin, or by serine protease inhibition with 3,4-dichloroisocoumarin. Both effects of thrombin and PMA on PCA in SW-480 cells were blocked by pretreatment of cells with cycloheximide or actinomycin D, indicating that the response required de novo protein and RNA synthesis. The thrombin-enhanced PCA depended on the activation of protein kinase C (PKC) as it was diminished by staurosporine and calphostin C. Moreover, stimulation of SW-480 cells by thrombin or PMA led to a significant increase in TF mRNA within 3 h as measured by the reverse-transcription PCR method, which was also dependent on the activation of PKC. The unaltered decay rate of thrombin-enhanced TF mRNA, evaluated after the addition of staurosporine, suggested that its inhibitory effect occurred at a transcription level. Our data suggest that thrombin enhances TF gene expression and protein synthesis in tumour cells in vitro via PKC activation. The induction of TF expression in tumour cells by thrombin indicates that tumour-associated PCA might have a positive-feedback effect on in vivo local propagation of thrombus by thrombin formation.