Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Background
The transmembrane Eph receptors (Eph) and their ephrin ligands represent the largest subfamily of receptor tyrosine kinases. Eph/ephrins are key players in cell-cell communication due to their capability of bidirectional signaling. There is evidence that Eph/ephrins also play an important role in tumor progression and metastasis. Since hypoxia is an important elicitor for metastatic behaviour of tumor cells, the aim of our study was to investigate the influence of hypoxia on Eph and ephrin expression in primary and metastatic melanoma cell lines.

Materials and methods
The influence of experimental hypoxia (6 to 72 h) on viability and metabolism of three melanoma cell lines (Mel-Juso, A375, and A2058) was characterized using MTT tests and cellular uptake of both ¹⁸F-fluoromisonidazole (FMISO) and ¹⁸F-fluorodeoxyglucose (FDG). The mRNA expression of EphA2, EphB4, ephrinA1 and ephrinB2 was analyzed with quantitative RT-PCR. Protein synthesis was determined by flow cytometry.

Results
The uptake of FMISO increased in all three melanoma cell lines after incubation under hypoxic conditions. The FDG uptake under hypoxic conditions decreased in all three cell lines. The MTT test demonstrated that viability of A375 cells decreased to 29±3% after 72 h of hypoxia. A2058 cells showed only a weak decrease of viability by approximately 30%, whereas viability of Mel-Juso cells under hypoxia was not influenced. In all cells Eph/ephrin gene expression under hypoxic and normoxic conditions showed only minor differences, except for EphA2 expression in A375 cells, which increased by >40% after 12 h hypoxia. Flow cytometry showed no alteration in ephrin ligands under hypoxic conditions. In contrast, after 72 h hypoxia we detected a slight increase in EphB4 protein in all melanoma cell lines, and enhanced EphA2 protein only in metastatic cell lines A375 and A2058.

Conclusion
The metastatic melanoma cell lines A375 and A2058 react more sensitive to hypoxic conditions than the primary melanoma cell line Mel-Juso. Experimental hypoxia increases Eph receptor gene expression and protein synthesis, particularly, in metastatic melanoma cell lines, which could be indicative for a further mechanism by which hypoxia affects tumor metastasis.