Low molecular weight heparin (lmwh) inhibits tumor growth through the sequestration of angiogenic cytokines

1071 BACKGROUND: Anticoagulation by the use of unfractionated or low molecular weight heparins (LMWHs) has been shown to lead to a survival advantage in cancer patients. There is compelling evidence that these are a result of non-coagulation pathways but the mechanism remains unclear. We hypothesize that soluble heparins inhibit angiogenesis by sequestering growth factors such as FGF away from the target cell surface. In this study we also identify the FGF molecular pathways involved in heparin-induced tumor endothelial cell inhibition as well as determination of the in vivo specific tumor inhibition across different LMWHs.
 METHODS: A murine cell line constitutively secreting FGF-1, named Clone C, was established by stably transfecting a FGF-1 expression construct into NIH 3T3 cells. Clone C cells were implanted into mice to yield a working in vivo model of a highly angiogenic tumor. MTS Cell Proliferation assay (Promega) was used to characterize cell growth patterns with heparin treatment in vitro. FGF chimeric protein fused to a hemagglutinin peptide tag at the carboxyl-terminus end was conjugated to a biotin antibody. This conjugate was subsequently bound to an avidin coated Crystalplex bead allowing microscopic tracking of the movement of FGF in the presence or absence of heparins. Whole cell lysates were prepared and protein was visualized by Western blotting.
 RESULTS: Clone C tumors in vivo growth were significantly inhibited by LMWHs. The control group tumors were grossly angiogenic and well vascularized. In marked contrast, the heparin treated tumors were smaller, pale and possessed only scant peri-tumoral vessels. Immunohistochemistry staining confirms significantly decreased angiogenesis in the LMWH group. No organ hemorrhage was evident on detailed tissue survey. Direct visual evidence of FGF sequestration occur concomitant with a decrease in Tumor Derived Endothelial Cell (TDEC) mitogenesis. LMWHs inhibit FGF-induced TDEC mitogenesis in a concentration and time dependent manner. The end result is a significant inhibition of down stream signaling through the extracellular signal-regulated kinases (ERK).
 CONCLUSIONS: LMWHs both strip and sequester FGF from its receptor resulting in a down regulation of receptor activity and tumor endothelial cell mitosis.