Protection Against Hydrogen Peroxide–Induced Cell Death and Nuclear Condensation in ARPE–19 Cells by Curcumin

Abstract

Purpose: : Several studies suggest that antioxidant–rich diets may protect against age–related macular degeneration (AMD), which is the leading cause of blindness in the elderly. Although the exact etiology of AMD is not known, the retinal pigment epithelium (RPE) is considered a primary target via oxidative cell damage. Data from in vitro studies showed that hydrogen peroxide (H₂O₂)–induced oxidative stress leads to RPE cell damage and death. Curcumin, an active phenolic compound extracted from turmeric, has been shown to possess strong antioxidant activity among many other pharmacological properties. The purpose of this study is to determine if curcumin protects RPE cells against H₂O₂–induced oxidative stress and cell death.

Methods: : Cell viability assay: cultured RPE cells (ARPE–19) were grown to sub–confluence followed by pretreatment with curcumin (25–100 µM) for 1 hr at 37 ºC. Cells were then washed and incubated with H₂O₂ (100–600 µM) in fresh medium for 24 hr. The percentage of viable cells was determined using trypan blue dye exclusion. Nuclear condensation, which is a hallmark of apoptosis, was determined using Hoechst 33342 staining and fluorescence microscopy. Cells were pretreated with curcumin (100 µM) for 1 hr and then the percentage of condensed nuclei were determined at 0, 2, 4, 6, 8, 10 and 24 hr after treatment with H₂O₂ (600 µM).

Results: : Treatment of ARPE–19 with H₂O₂ (100–600 µM) resulted in dose–dependent cell death (52% at 500 µM H₂O₂ and 86% at 600 µM H₂O₂) and resulted in an increase in the number of cells containing condensed nuclei in a time–dependent manner (600 µM H₂O₂: 15% at 4 hr and 46% at 24 hr). Pretreating cells with curcumin for 1 hr prior to insult notably attenuated cell death with 600 µM H₂O₂: 86%, 73%, 66% and 41% with 0, 25, 50 and 100 µM curcumin, respectively. Furthermore, curcumin pretreatment (100 µM) decreased the percentage of cells containing condensed nuclei with 600 µM H₂O₂: 3% at 4 hr and 14% at 24 hr.

Conclusions: : The results of this study indicate that curcumin provides substantial protection against hydrogen peroxide–induced cell death and nuclear condensation in ARPE–19 cells and may therefore, be useful as a potential agent in the treatment of disorders associated with oxidative stress–induced cell damage, in particular AMD.

Keywords: antioxidants • apoptosis/cell death • retinal pigment epithelium