X-ray irradiation influences Eph receptors and cellular properties in human melanoma cells

Introduction: There is experimental evidence that X-ray irradiation influences survival and metastatic properties of tumor cells. On the other hand, metastasis and cellular motility can be modified by EphA2 and EphA3, two members of the Eph receptor/ephrin family of receptor tyrosine kinases. The aim of this study was to analyze whether there is a molecular link between X-ray irradiation, Eph expression, and modification of metastasis-associated cell properties in human melanoma cells.
Material and Methods: We irradiated one pre-metastatic and three metastatic human melanoma cells lines, including one self-generated metastatic variant, with X-rays (5 and 10 Gy). At day 1 and day 7 post irradiation (p.i.) we analyzed cell proliferation, colony formation, adhesion, and migration. Additionally, selected Eph receptors and ephrin ligands were analyzed regarding irradiation-dependent changes in mRNA and protein content. For EphA2 and selected downstream signaling molecules we determined the phosphorylation status, representing protein activity.
Results and Discussion: Irradiation resulted in decreased proliferation and colony formation. Colony formation showed partial recovery at 7 days p.i. with 5 Gy. Regarding cell adhesion, we detected an irradiation-induced increment paralleled by a decrease in migration of Mel-Juso and Mel-Juso-L3 cells and, in part, A375 cells. Thus, we assume that X-rays merely act anti-metastatic on the investigated melanoma cells. Expression of the ephrins A1 and A5 generally was very low and after Xray showed a substantial decrement for ephrin A5 in all cells, but a heterogenous behaviour for ephrin A1. For EphA2 we detected a decrease after irradiation both in expression and activity at 7 days p.i. In contrast, EphA3 was found to be up-regulated in 3 of 4 analyzed cell lines, raising the question, if there is a counter-regulation between EphA2 and EphA3. Analyzing downstream signaling, we detected decreased Src kinase and focal adhesion kinase (FAK) phosphorylation in A375, A2058, and Mel-Juso cells at 10 Gy for Src and both 5 Gy and 10 Gy for FAK at 7 days p.i.
Conclusion: Our findings indicate that irradiation-induced downregulation of EphA2 and up-regulation of EphA3 in human melanoma cells is associated with anti-metastatic effects. The observed effects are assumed partly to be mediated by regulation of Src and FAK through EphA2.