Antineoplastic agent busulfan regulates a network of genes related to coagulation and fibrinolysis

Abstract

Purpose Hepatic veno-occlusive disease (HVOD) is one of the major complications following hematopoietic stem cell transplantation (HSCT). Although high-dose busulfan is associated with the development of HVOD, the underlying molecular mechanisms are still unknown.Methods Transcriptional gene regulation by busulfan was profiled using Affymetrix GeneChip® Human Genome U133 Plus 2.0 arrays. Messenger RNA (mRNA) expression of regulated genes was assessed by TaqMan real-time polymerase chain reaction (PCR), and protein expression and secretion was determined by enzyme-linked immunosorbent assay (ELISA)in cell supernatants, lysates, and patient plasma.Results Plasma levels of plasminogen activator inhibitor(PAI)-1 significantly increased 48 h after starting busulfan treatment IV in children preconditioned for HSCT. In vitro,busulfan significantly induced plasminogen activator inhibitor-1 (PAI-1) expression in endothelium-like ECV304 cells in a concentration- and time-dependent manner. Comparative transcriptional profiling of busulfan-treated and control ECV304 cells identified differential expression of genes related to coagulation and fibrinolysis, including tissue factor, tissue factor pathway inhibitor-1, protein S, thrombospondin-1, urokinase receptor, and PAI-1, as well as activin A and transforming growth factor beta 1 (TGF-β1). Ingenuity pathway analysis (IPA) suggested TGF-β1 as a central modulator of gene regulation by busulfan. Consequently, expression of tissue factor, urokinase receptor, and PAI-1 mRNA and PAI-1 protein secretion induced by busulfan were significantly reduced by the activin A/TGF-β1 inhibitor SB 431542 in ECV304 and primary endothelial cells.Conclusions This is the first report that directly relates busulfan exposure to antifibrinolytic activity by PAI-1 and hypercoagulation possibly mediated by members of the TGF-β1 family. This suggests further research to evaluate activin A and TGF-β1 as potential targets for HVOD treatment.

Polymorphism in TGFB1 is associated with worse non-relapse mortality and overall survival after stem cell transplantation with unrelated donors

“…21 However, this remains unclear. Interestingly, a recent report 22 has shown evidence of a role for TGF-β1 in limiting both the growth and function of the thymic medulla, another potential niche for its influence on the outcome of HSCT.…”

Pulmonary hypertension associated with busulfan

“…This drug might induce a hypercoagulation state mediated by members of TGF-ß1 family and antifibrinolytic activity through plasminogen activator inhibitor (PAI)-1. 17 Moreover, busulfan induces an increase in the numbers of circulating endothelial cells and their progenitors. 18 In addition, the renewal of endothelial cell upon the occurrence of endothelial dysfunction appears to depend on resident progenitor cells, without the therapeutic effect of bone marrow transplantation.…”

Role of Pharmacogenetics in Hematopoietic Stem Cell Transplantation Outcome in Children

“…Defibrotide does not have a direct anticoagulant function, but shows pleiotropic antithrombotic properties acting on the fibrinolytic pathway: it enhances the expression of tissue plasminogen activator (tPA), a serine protease that catalyzes the conversion of plasminogen to plasmin; it enhances the activity of plasmin itself and reduces the level of plasminogen activator inhibitor type-1 (PAI-1) [ 68 ]. Interestingly, plasma levels of PAI-1 have been shown to significantly increase 48 h after starting the first BU infusion in 18 children preconditioned for HSCT, and genome-wide transcriptional profiling of BU-treated and -untreated cells revealed a differential expression of genes related to the coagulation system modulated by TGF-β1, rather than TNF-α [ 69 ]. These results give rationale to the clinical practice of combining defibrotide in a BU-based conditioning regimen.…”