Alpha-eleostearic acid (alpha-ESA, 9Z11E13E-18.3). a linolenic acid isomer with a conjugated triene system. is a natural and biologically-active Compound that has been shown to possess potent anti-tumor properties Herein, we demonstrate alpha-ESA induced apoptosis and autophagy with reactive oxygen species (ROS) generation in HeLa cells Treatment with alpha-ESA caused inhibition of phosphorylated (p)AKT and elongated the sub G1 phase in the cell cycle. indicating induction of apoptosis. Autophagy was also induced by alpha-ESA treatment, causing low pAKT and pP70S6K activities, increasing pERK1/2 and leading to a higher conversion rate of LC3 I to LC3 II compared to that of the control. The autophagy was further confirmed by fluorescence microscopy and flow cytometry through monodansylcadavarine (MDC) staining It appears that the role of autophagy is a protective mechanism against cell death in alpha-ESA-treated HeLa cells. Subsequently, we found that treating HeLa cells with alpha-ESA induced the generation of reactive oxygen species (ROS) The phosphorylation of P70S6K. downstream of mTOR signaling, and AKT were further reduced by pretreatment with N-acetyl-L-cysteine (NAC), an ROS scavenger, whereas the phosphorylation of ERK1/2 and the conversion of LC3 I to LC3 II were further enhanced As a result, the blocking of the action of ROS promoted alpha-ESA-induced apoptosis and autophagy. Taken together, our results indicate that the generation of ROS by alpha-ESA treatment impedes the progress of apoptosis and excessive autophagy formation which takes part in cell death, thus impeding death promotion. (C) 2009 Elsevier Inc All rights reserved