Inhibition of uveal melanoma cell proliferation by nepafenac, a selective cyclooxygenase–2 (COX–2) inhibitor

Abstract

Abstract: : Purpose: The expression of COX–2 in uveal melanoma, the most common malignant intraocular tumor in adults, has been linked to markers of poor prognosis. COX–2 expression induces a variety of cytokines that can alter cellular proliferation, invasion, and apoptotic processes. The aim of this study is to examine the effect of nepafenac, a selective COX–2 inhibitor, on the proliferative rate of five human uveal melanoma cell lines with COX–2 expression and the same five cell lines without COX–2 expression. Methods:Five human uveal melanoma cell lines (92.1, SP6.5, MKT–BR, OCM–1, and UW–1) were transfected with COX–2 cDNA inserted into the pcDNA3 vector. The cells were grown to confluence in RPMI 1640 geneticin selective medium supplemented with 5% Fetal Bovine Serum. COX–2 expression in the transfected cell lines was verified by Western blot analysis. Non–transfected cells from the same five cell lines were used as controls. The sulforhodamine–B assay was used to compare uveal melanoma cell growth with and without amfenac, the active metabolite of nepafenac, as per the National Cancer Institute protocol. Briefly, all transfected and non–transfected cell lines were seeded at a concentration of five thousand cells per well in two ninety–six well plates. Amfenac at the recommended IC₅₀ of 150 nM was added to the wells in one plate. Cells were allowed to incubate for 48 hours and then total cellular protein content per well was assessed. The averaged results per COX–2 transfected and non–transfected uveal melanoma cell lines were recorded. The Student’s t– test was used to compare results from the cells cultured with and without amfenac. Results: A statistically significant difference in total cellular content was observed between all cells treated with amfenac and the same cell lines without amfenac. A decrease in total cellular content, correlating with decreased cellular proliferation, was seen not only in the cells transfected to express COX–2 but also in the cells that do not express COX–2 in vitro. Conclusions: The selective anti–COX–2 molecule amfenac inhibited proliferation of all tested uveal melanoma cell lines. Both the COX–2 expressing cell lines and the non–transfected cell lines showed a decrease in proliferation, which indicated that amfenac not only functioned by inhibiting COX–2 expression, but also functioned through a second unknown pathway. Further trials should be undertaken to study the effect of selective COX–2 inhibitors on uveal melanoma.

Keywords: melanoma • drug toxicity/drug effects • proliferation