Tissue factor/FVIIa activates Bcl-2 and prevents doxorubicin-induced apoptosis in neuroblastoma cells

• Type of Material: Article
• Authors: Ju Fang, Emory University; Lubing Gu, Emory University; Ningxi Zhu, Emory University; Hao Tang, Wuhan No.1 Hospital; Carlos S Alvarado, Emory University; Muxiang Zhou, Emory University
• Language: English
• Date: 2008
• Publisher: BioMed Central
• Publication Version: Final Published Version
• Copyright Statement: © 2008 Fang et al; licensee BioMed Central Ltd.
• License: Creative Commons Attribution 2.0 Generic
• Final Published Version (URL): http://www.biomedcentral.com/1471-2407/8/69
• Title of Journal or Parent Work: BMC Cancer
• ISSN: 1471-2407
• Volume: 8
• Issue: 69
• Grant/Funding Information: MZ also provided financial support for the study.; This work was supported by grants from the NIH (R01 CA123490), the Leukemia and Lymphoma Society (6249-05, and 6033-08), CURE Childhood Cancer, and Hope Street Kids (HSK).
• Research Categories: Health Sciences, General; Health Sciences, Oncology; Health Sciences, Obstetrics and Gynecology

Abstract

Background Tissue 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. Methods Gene 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. Results Enforced 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. Conclusion This 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.