Influence of irradiation on para- and autocrine regulation of extracellular S100A4 (metastasin) and its receptor RAGE in B16 mouse melanoma cells

Background:

Malignant melanoma is one of the most invasive and metastatic tumors. A common therapeutic approach towards metastases will combine radiation with chemotherapy and/or surgery. The interaction between tumor and inflammatory cells, e.g., via S100A4 (metastasin) and the receptor for advanced glycation endproducts (RAGE), is hypothesized to play a key role in metastasis of melanoma. In this study the contribution of para- and autocrine S100A4-RAGE activation to growth, motility and migration of metastatic melanoma and inflammatory cells before and post irradiation was investigated.

Materials and methods:

Mouse melanoma cells (B16), macrophages (RAW; a model for tumor associated macrophages (TAM)) and B16/RAW cocultures (ratio 1 to 5) were exposed to single dose irradiation (5, 10, and 20 Gy, compared to sham-irradiated controls) for 0, 3 and 6 days. S100A4 and RAGE expression in these cells was quantified via real-time RT-PCR, Western-blot analysis and immunochemistry. Cell growth and cellular viability was detected by MTT assay. Migration assays of non- and irradiated cells were performed with and without chemoattractants (supernatants of irradiated cocultures after 6 days). Additionally, the actin cross-linker L-plastin was investigated as a migratory marker.

Results:

Post irradiation, S100A4 and RAGE mRNA expression was significantly increased in B16 and RAW cells but not in cocultivated cells. S100A4 protein expression was only detected in irradiated B16 cells whereas RAW cells always showed high levels in non- and irradiated cells. Interestingly, cocultures showed only minor S100A4 expression levels with a further reduction of S100A4 after irradiation. In contrast, RAGE protein showed only slight differences. A significant reduction of cell viability was observed after irradiation via MTT assay. On the other hand, migratory activity was significantly increased in B16 and cocultures after irradiation whereas RAW cells showed a significant decrease. Furthermore, chemoattractants significantly induced the migration in non-irradiated B16 cells.

Conclusion:

Irradiation of both melanoma cells and macrophages alters their migratory and invasive activity. Under conditions of cocultivation these effects were more pronounced. We suppose an involvement of para- and autocrine regulation of extracellular S100A4 and its receptor RAGE in melanoma cells and TAM, thereby changing functional properties of melanoma cells towards a promigratory phenotype.