Hypoxia Regulates Eph/Ephrin-Facilitated Adhesion and Proliferation Of Chronic Myeloid Leukemia Cells

Recently, hypoxia has been demonstrated to be correlated with metastasis. The cellular response to oxygen deprivation involves a series of metabolic and biosynthetic events associated with adaptation to a hypoxic environment. Meanwhile, eph receptors and their ligands (ephrins) have also been proved to play a very important role in regulating the migrationand proliferationof leukemia cell in our early study. Therefore, we performed the experiment to address the hypothesis that hypoxia might be an important mediator linking Ephs/ephrins expression to chronic myeloid leukemia cell adhesion and proliferation.

K562 cells were cultured in a hypoxia environment with different density of oxygen (1%, 5%, 20%). The expression of HIF-1α, EphA5, EphB4, EfnA1, EfnB2 and signaling pathway proteins like p-FAK, p-Src, p-Paxilin, p-p130, p-cofilin, p-Akt, cyclin D3, cyclin E, p-Rb of K562 cells in different hypoxia environment were determined by real-time PCR and Immunoblot analysis. The K562 cells adhesion to endothelial cells and bone marrow stromal cells were tested with the adhesion assay kit. K562 cell proliferation, apoptosis and cell cycle were also detected with immunofluorescence analyses. After knocked down HIF-1α gene in K562 cells, the related protein levels and adhesion ratio were determined by the same methods.

The results showed that the mRNA levels of HIF-1α were enhanced after 10 h of hypoxic exposure (1% O₂), compared with normal exposure (P<0.05), as well as the mRNA levels of EphA5, EphB4, EfnA1, EfnB2 and related signaling pathway proteins were enhanced after 24h of hypoxic exposure (P<0.05). The protein levels of HIF-1α, EphA5, EphB4, EfnA1, EfnB2 and signaling pathway proteins like p-FAK, p-Src, p-Paxilin, p-p130, p-cofilin, p-Akt, cyclin D3, cyclin E, p-Rb also increased after 24h of hypoxic exposure (1% O₂). After knocked down HIF-1α, the expression of Eph/ephrin didn’t enhance after 72h of hypoxic exposure(1% O₂) ,which proved HIF-1α regulate Eph/ephrin expression. The adhension to endothelial cells was significantly up-regulated (1.6 to 2-fold, P<0.05) after 36 h of hypoxic exposure (1% O₂), the apoptosis cells down-regulated (1.3 to 1.5 fold, P<0.05).

We concluded that Eph receptors and ephrins were closely correlated with chronic myeloid leukemia cell adhesion and proliferation, and this process was regulated by hypoxia. Our findings shed new light on the regulation of this intriguing receptor/ligand family and present evidence for the role of Ephs/ephrins in chronic myeloid leukemia cells.

No relevant conflicts of interest to declare.