Abstract
BackgroundTissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines.MethodsGene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively.ResultsEnforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2.ConclusionThis study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF-FVIIa pathway may contribute, at least in part, to chemotherapy resistance in neuroblastoma.
Highlights
Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade
TF expression and sensitivity to doxorubicin in neuroblastoma cell lines We have examined TF protein in two neuroblastoma cell lines and found that the level of TF expression was remarkably variable among the different cell lines
B, TF-transfected SK-N-SH cells were treated with different concentrations of either STAT5 siRNA or control siRNA for 24 h
Summary
Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Tissue factor (TF) is a transmembrane protein that belongs to the class II cytokine receptor superfamily that shares a significant degree of homology with the interferon gamma receptor [1] It binds to coagulation factor VII (FVII) and its active form (FVIIa), initiating the coagulation cascade via the extrinsic pathway. In addition to its role in coagulation, accumulating evidence suggests that TF regulates intracellular signaling pathways [2], that play a crucial role in embryonic development [3], inflammation [4], angiogenesis [5,6], and tumor development and metastasis as well [6,7] These latter two processes are mediated through activation of the Src family – which subsequently activates PI3K/Akt and p38 MAPK pathways to positively regulate tumor cell growth [8,9] and PAR-2 activation as well – which results in increased cell migration [10,11]. Specific interaction of the cytoplasmic domain of TF with actin-binding protein-280 (ABP-280) has been shown to mediate tumor cell metastasis and vascular remodeling in human bladder carcinoma cells [13]
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