Alpha-eleostearic acid induces autophagy-dependent cell death through targeting AKT/mTOR and ERK1/2 signal together with the generation of reactive oxygen species

Abstract

Alpha-eleostearic acid (α-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 α-ESA induced apoptosis and autophagy with reactive oxygen species (ROS) generation in HeLa cells. Treatment with α-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 α-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 α-ESA-treated HeLa cells. Subsequently, we found that treating HeLa cells with α-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 α-ESA-induced apoptosis and autophagy. Taken together, our results indicate that the generation of ROS by α-ESA treatment impedes the progress of apoptosis and excessive autophagy formation which takes part in cell death, thus impeding death promotion.