Abstracts

Rationale: Several studies have shown apoptotic and anti-tumoral effects of valproate (VPA). Levetiracetam (LEV) is a promising, new antiepileptic drug with positive reports regarding both effects and side-effects. In a recent study in porcine ovarian cells, LEV, as opposed to VPA, did not have any apoptotic effects. The aim of the present study was to investigate whether LEV exerts apoptotic effects in human ovarian cells, and to compare the results with VPA. Methods: Granulosa cells were obtained from ovaries of women between 25-40 years old who underwent laparoscopic procedures of ovarian drilling due to infertility. Cells were cultured for 48 hrs in the presence of 1x10-7 M of testosterone and different concentrations of LEV (12, 20, 50 or 80 μg/ml) or VPA (75, 100, 250, or 350 μg/ml) with and without FSH. After 48 h of culture, lysates were incubated with the caspase-3 substrate Ac-DEVD-Pna. After 1.5 hrs, the absorbance of the lysate was measured at 405 nm using a micro-ELISA plate reader. Results: In both untreated and FSH-stimulated cells, LEV in concentrations of 20, 50 and 80 µg/ml, and VPA in concentrations of 100, 250 and 350 µg/ml, significantly increased caspase-3 activity. In both situations, the pro-apoptotic effect of LEV was more pronounced than that of VPA. Maximum increase in caspase-3 activity was around 100% for LEV and slightly above 10% for VPA under both basal conditions and after FSH stimulation. The effect was not concentration-dependent for any of the drugs. Conclusions: Both LEV and VPA significantly increased caspase-3 activity with LEV being the most potent. Caspase-3 activity measurement is a simple and quantitative technique to measure apoptosis. Caspase-3 is activated at a point of convergence for the intrinsic and extrinsic apoptosis induction pathways, so its activity should give a reliable measure of ongoing levels of apoptosis. We therefore conclude that both drugs act as apoptotic agents in ovarian cells and that the pro-apoptotic effect of LEV was more pronounced than that of VPA. The effect of LEV even at therapeutic drug concentrations should be verified in other models of apoptosis.