In vitro effects of preserved and unpreserved antiglaucoma drugs on apoptotic marker expression by human trabecular cells

Background: The mechanisms of trabecular cell loss in glaucoma patients are poorly understood. In order to determine whether drug-induced apoptosis could be one of the mechanisms by which trabecular cells die in glaucoma, we evaluated the effect of benzalkonium-preserved (BAC+) or preservative-free (BAC-) antiglaucoma medications on apoptotic marker expression by cultured human trabecular meshwork (HTM) cells. Methods: Normal and glaucomatous trabecular cell lines were treated for 15 min with antiglaucoma drugs (1/100 and 1/10 dilutions): timolol BAC+ or BAC-, betaxolol BAC+ or BAC-, latanoprost BAC+ or pure BAC. Apo2.7 expression, annexin V binding and DNA content were evaluated by flow cytometry and confocal microscopy. Results: Results obtained in the two cell lines were similar for all tested drugs and criteria. In a 1/100 dilution, unpreserved beta-blockers had no apoptotic effect, preserved beta-blockers and latanoprost significantly increased Apo2.7 expression only, while BAC significantly increased all three apoptotic markers. When tested in a 1/10 dilution, all drugs except unpreserved timolol triggered a 2- to 3.5-fold increase in apoptotic features, whereas up to 95% of the cells underwent apoptosis upon treatment with BAC (representing a 9-fold increase over the background level). Conclusion: At concentrations higher than those supposed to be found in the aqueous humor after instillation (1/100 dilution), unpreserved beta-blocker exhibited no proapoptotic activity on HTM cells in vitro. Benzalkonium-containing beta-blockers and prostaglandin analogue triggered mild expression of one out of three apoptotic markers, while the pro-apoptotic effect observed with BAC appeared to be largely hindered by active compounds in the preserved eyedrops.