Caffeic acid phenethyl ester blocks apoptosis induced by low potassium in cerebellar granule cells

Abstract

Primary cultures of cerebellar granule neurons (CGNs) were prepared from 8-day-old Wistar rats, and maintained in an appropriate medium containing a high (25mM) concentration of KCl. To induce apoptosis, culture medium was replaced with serum-free medium (containing 5mM KCl) 8 days after plating. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling (TUNEL) method, and by flow cytometry. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by low K+ (5mM) concentrations, the potential anti-apoptotic effect of caffeic acid phenethyl ester (CAPE), a potent flavonoid antioxidant, was tested in this experimental model. It was found that CAPE (10μg/ml) promoted cell survival and was capable of blocking the apoptotic process as assayed by both TUNEL and flow cytometric methods. The same concentration of CAPE prevented the formation of ROS induced by low K+. Since there is evidence that low K+-induced apoptosis in CGNs is associated with a drop in intracellular Ca2+ concentration ([Ca2+]i), activation of the cell death effector proteases caspase-3 and caspase-9, and of the transcription factor nuclear factor kappa B (NF-κB), the interference of CAPE with these purported mediators of apoptosis was also evaluated. It was found that CAPE did not interfere with the marked decrease in [Ca2+]i induced by low K+, whereas it completely blocked caspase-3, caspase-9, and NF-κB activation. It is concluded that CAPE could exert its anti-apoptotic effect in CGNs by blocking ROS formation and by inhibiting caspase activity.