Background. EphB4 receptor tyrosine kinase is of diagnostic and therapeutic value due to its overexpression in breast tumors. Dual functions of tumor promotion and suppression have been reported for this receptor based on presence or absence of its ligand. To elucidate such discrepancy, we aimed to determine the effect of time- and dose-dependent stimulation of EphB4 on viability and invasion of breast cancer cells via recombinant ephrinB2-Fc. Methods. Cells were seeded into multiwell plates and were stimulated by various concentrations of preclustered ephrinB2-Fc. Cell viability was measured on days 3 and 6 following treatment using alamar-blue when cells were in different states of confluence. Results. Stimulation of cells with ephrinB2 did not pose any significant effect on cell viability before reaching confluence, while inhibition of cell growth was detected after 6 days when cells were in postconfluent state following a dose-dependent manner. EphrinB2 treatment did not affect tubular formation and invasion on matrigel. Conclusion. This study showed that EphB4 can differentially inhibit cells at post confluent state and that presence of ligand manifests growth-inhibitory properties of EphB4 receptor. It is concluded that growth inhibition has occurred possibly due to long treatment with ligand, a process which leads to receptor downregulation. 1. Introduction Cancer cells express many factors that enable them to overcome regulatory barriers towards uninterrupted growth and to survive in abnormal circumstances in which normal cells will not [1]. Among such factors, tyrosine kinases play pivotal roles. Eph (Erythropoietin Producing Hepatoma) receptors comprise the largest family of tyrosine kinases and are divided into two classes of EphAs and EphBs based on their sequence homology. Receptor activation occurs via their membrane bound ligands ephrinAs and ephrinBs, respectively [2]. One distinguishing feature of this family is their ability to propagate bidirectional signals in cells expressing receptor and ligand; in other words, both Eph receptors and ephrin ligands are capable of inducing downstream signals in the form of forward and reverse signals [3]. Owing to such spatial orientation, Eph receptors and ephrin ligands construct a communication system between cells expressing the receptor and ligand, which allows them to be involved in many physiological processes such as proliferation, morphology and movement, normal organ out-growth during neuronal path finding, and angiogenesis [4–7]. Recently, their involvement in abnormalities such as cancer
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