Activation Of EphrinB2/EphB4 Influences Myeloid Leukemia Cell Migration and Invasion

Eph receptors and ephrin ligands are cell-surface molecules capable of bidirectional signaling that control cell-cell interactions, migration and invasion. However, their role and regulation in myeloid leukemia cells remain to be defined. To address the hypothesis that Ephrin/EphB is an important regulator of myeloid leukemia cell migration and invasion, we first screened the mRNA levels of 23 eph and ligand ephrin RTK family members in myeloid leukemia cells (K562, HL-60, THP-1) and mononuclear cells from healthy donors, then found that EphB4, EphA5, EfnA1 highly expressed in most myeloid leukemia cells compared to healthy donors(P<0.05). Both the mRNA and protein levels of EphB4 and EphA5 were detected in 13 primary myeloid leukemia cells (5 from patients with extramedullary leukemia among 13 cases) and 10 mononuclear cells from healthy donors by real-time RT-PCR and Immunoblot analysis. The results showed that both the mRNA and protein levels of EphB4 and EphA5 were higher in 13 primary myeloid leukemia cells relative to the 10 healthy donors (P=0.046). Moreover, the EphB4 were highly expressed in 5 patients with extramedullary leukemia compared with 8 patients without extramedullary leukemia. These findings indicated that EphB4 and EphA5 expression were correlated with the development of myeloid leukemia cells, moreover, EphB4 may be closely related with myeloid leukemia cell migration or invasion. To further clarified the question, migration were determined in leukemia cell lines (K562 cells) which were treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins by transwell migration assay. Invasion were also determined by matrigel invasion assay. The results showed that, after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were significantly enhanced compared to Fc proteins stimulation (1.8 to 2.5-fold, P<0.05), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.2 to 1.8-fold, P<0.05). However, after ephrinA1–Fc stimulation, the numbers of K562 cells migrating through transwell chamber didn’t significantly increase compared to Fc proteins stimulation (P>0.05), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P>0.05). These findings indicated that ephrinB2–Fc could activate EphB4, leading to the change of myeloid leukemia cell migration and invasion. Further study may help to assess a promising potential of this protein to be used as a prognostic marker or as a target for a molecular therapy.