The Cyclooxygenase–2 Inhibitor Nepafenac Improves the Function of Macrophages Suppressed by Uveal Melanoma Conditioned Medium

Abstract

Abstract: : Purpose: Cyclooxygenase–2 (COX–2) expression has been linked to poor prognosis in uveal melanoma. The aim of this study was therefore to investigate macrophage function upon exposure to media from COX–2 and non–COX–2 transfected uveal melanoma cells. In addition, we aimed to investigate the effect of a COX–2 inhibitor on macrophage function following exposure to uveal melanoma conditioned media. Methods: Five human uveal melanoma cell lines (92.1, MKT–BR, OCM–1, SP6.5 and UW–1) with and without COX–2 transfection, as well as one monocyte cell line (28SC), were seeded in 12 well plates at a concentration of 1 x 10⁶ cells/ml. After 18 hours, 28SC were exposed to melanoma–conditioned medium with and without amfenac, the active metabolite of nepafenac, at the recommended IC₅₀ of 150 nM. 28SC incubated in fresh medium was used as control. 24 hours after media transfer, 28SC were stimulated with IFN–gamma and LPS. The experiment was done in triplicate. 28SC nitric oxide (NO) production was determined using the Greiss reaction. The Student’s t– test was used to compare results of 28SC incubated with conditioned medium from COX–2 transfected versus non–COX–2 transfected melanoma cell lines as well as 28SC incubated with conditioned medium from these cell lines with and without amfenac. Results: Compared to control medium, conditioned medium from all uveal melanoma cell lines caused a statistically significant decrease in macrophage NO production. Different effects were seen with the medium from different uveal melanoma cell lines (UW–1>OCM–1>MKT–BR>92.1>SP6.5). Compared to control medium from non–COX–2 transfected uveal melanoma cell lines, medium from COX–2 transfected uveal melanoma cell lines caused a significant decrease in macrophage NO production (OCM–1>UW–1>MKT–BR>92.1>SP6.5). Addition of the COX–2 inhibitor significantly decreased the amount of macrophage NO production suppression induced by conditioned medium from all uveal melanoma cell lines. Conclusions: These results indicate that soluble factors produced by uveal melanoma cell lines can significantly inhibit NO production by macrophages, decreasing their cytotoxic ability. The addition of a COX–2 selective inhibitor significantly abates the inhibition of NO production by the macrophages. Three of the five transfected cell lines gave more inhibition compared to the same non–transfected cell line; implying that COX–2 may play a role in this immunemodulation.

Keywords: melanoma • melanocytes