Overexpression of tissue factor pathway inhibitor in CHO-K1 cells results in increased activation of NF-κB and apoptosis mediated by a caspase-3 independent pathway
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There is now circumstantial evidence that tissue factor pathway inhibitor (TFPI) is not only a major anticoagulant, but also has proapoptotic properties. The current study was designed to address the role of TFPI on signalling pathways and apoptosis. The non-TFPI expressing cell line CHO-K1 was stably transfected with pcDNA3.1/V5-His-TOPO-TFPI and control cells were established by transfecting the CHO-K1 cells with pcDNA3.1/V5-His-TOPO. Sodium butyrate (NaBut) has been shown to induce the expression of recombinant proteins. Here we have used NaBut to increase the expression of TFPI as assessed by qRT-PCR and ELISA. Compared to the control cells, TFPI induced apoptosis in a concentration dependent manner as measured by a cell death detection assay. Independent of caspase-3 activation an increased cleavage of PARP was detected in the TFPI expressing cells. This was accompanied by downregulation of Bcl-XL, elevated levels of Bax, and increased translocation of the apoptosis initiating factor. Increased DNA binding activity of NF-κB was revealed by electrophoretic mobility shift assay when the TFPI level was elevated by NaBut together with an increased translocation of the NF-κB subunit p65. The results indicate that TFPI affected the apoptotic activity through a process independent of caspase-3, and was also able to increase the activation of the NF- κB pathway.
KeywordsTissue factor pathway inhibitor CHO-K1 cells Apoptosis NF-κB Anti-cancer Na-butyrate
The authors are grateful for the technical assistance of Erle Solheim. The study was financially supported by Grants from the Norwegian South-Eastern Health Authority.
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