Fang Jun, Gu Lubing, Zhu Ningxi, Tang Hao, Alvarado Carlos S, Zhou Muxiang
Division of Hematology/Oncology, Department of Pediatrics, Emory University School of Medicine, Atlanta, USA.
BMC Cancer. 2008 Mar 6;8:69. doi: 10.1186/1471-2407-8-69.
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.
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.
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.
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.
组织因子（TF）是一种跨膜蛋白，作为活化凝血因子VII（FVIIa）的受体，启动凝血级联反应。最近的研究表明，肿瘤来源的TF的表达还介导与肿瘤生长和凋亡相关的细胞内信号传导。我们目前的研究探讨了TF与FVIIa之间的相互作用调节神经母细胞瘤细胞系化疗耐药性的可能机制。
基因和小干扰RNA（siRNA）转染分别用于在TF阴性的神经母细胞瘤细胞系中增强TF表达，并在TF过表达的神经母细胞瘤细胞系中沉默内源性TF表达。通过蛋白质免疫印迹法检测基因转染细胞或用JAK抑制剂和LY2940０2处理的细胞中TF、Bcl-2、信号转导和转录激活因子5（STAT5）和蛋白激酶B（Akt）的表达以及STAT5和Akt的磷酸化。通过克隆形成试验测定肿瘤细胞生长。分别通过WST试验和膜联蛋白V染色（流式细胞术）分析阿霉素对神经母细胞瘤细胞系的细胞毒性和凋亡作用。
在存在FVIIa的情况下，在TF阴性的神经母细胞瘤细胞系中强制表达TF可诱导Bcl-2上调，导致对阿霉素耐药。相反，使用siRNA抑制TF过表达的神经母细胞瘤细胞系中的内源性TF表达导致Bcl-2下调，并对阿霉素诱导的凋亡敏感。此外，用FVIIa处理的表达高水平内源性或转染TF的神经母细胞瘤细胞容易使STAT5和Akt磷酸化。使用选择性药理抑制剂，我们证明JAK抑制剂I可阻断TF/FVIIa诱导的Bcl-2上调，而PI3K抑制剂LY2940０2则不能。
本研究表明，在神经母细胞瘤细胞系中，过表达的TF与FVIIa结合通过JAK/STAT5信号通路导致Bcl-2表达上调，从而产生抗凋亡作用。我们推测，这种TF-FVIIa途径可能至少部分地导致神经母细胞瘤的化疗耐药。
