As summarized in Fig autophagy up regulation in

As summarized in Fig. 1, autophagy up-regulation in terms of Beclin 1 induction, increase of LC3 II and p62 down-regulation were observed between 24 and 72 h of cell incubation, this coinciding with the end of the period 27-OH caused slight reduction of cell viability and proliferation as previously reported [19]. Moreover, the decrease of autophagy induction, as marked by the induction of p62 between 4 up to 7 days oxysterol treatment was consistent with the interval considered the pro-apoptotoic effect of 10 μM 27-OH was demonstrated, suggesting that oxysterol activates cytoprotective autophagy which provides sustained cellular survival (Supplementary Fig. 1). In addition, as depicted in Fig. 3, blockade of autophagy subsequently enhanced the apoptosis in U937 promonocytic cells. Using chemical and molecular approaches, Bafilomycin A1 and Beclin 1 siRNA administration respectively, we found that induction of autophagy was obligatory for low concentrations of 27-OH-induced survival response, most likely an adaptive survival strategy that recycles membrane transport and eliminates damaged organelles, to protect against the apoptotic cell death. This observation is consistent with previous work showing that 7-K induces NADPH oxidase-mediated autophagy as a cellular protective response that extenuated endoplasmic reticulum stress/apoptosis pathway induced by 7-K in human aortic SMCs [47]. An additional study shows that autophagy induction by 7-oxysterols, mixed in an atheroma-relevant proportion reduced 7-oxysterols induced cell death by reducing lysosomal membrane permeabilization where induced autophagy may have a cytoprotective effect to limit necrotic core formation in atheroma progression [48]. On the other hand, treatment of 158 N murine oligodendrocytes with a relatively high concentration (50 μM) of 7-K was shown to trigger a type of death termed oxyapoptophagy with the concomitent induction of autophagy and apoptosis [49]. Not only oxysterols but also low levels of oxLDLs, were demonstrated to activate autophagic markers in vascular cell lines whereas high concentrations of oxLDLs induce apoptosis by modulating oxLDL scavenger receptor 1 [50]. It has to be pointed out that the outcome of an autophagic response depends on oxidant concentration and exposure time. In fact, these oxidized lipids are able to trigger pro-survival autophagy upon treatment of cells only with low amount. In the last years, it has been accepted that serine/threonine kinases involving AMP-activated protein kinase (AMPK), mTOR, protein kinases C (PKCs), Akt and mitogen-activated protein kinases (MAPKs), such as ERK, p38 and c-Jun NH2-terminal kinase (JNK), have regulatory role in various steps of autophagy [51]. Indeed, these kinases either positively or negatively regulate autophagic response depending on the inducer and the cellular context [15]. In a recent study it has been shown that HNE, a lipid peroxidation-derived aldehyde, stimulated pro-survival autophagic response interms of LC3 II formation in a JNK-dependent mechanism in rat aortic SMCs [52]. Our results demonstrate that ERK and Akt phosphorylation is required for autophagy induction and also p62 up-regulation in 27-OH-treated U937 cells. Indeed, as shown in Fig. 2, pharmacological inhibition of these pathways by the MEK/ERK inhibitor PD98059 and the PI3K/Akt inhibitor LY294002 respectively, prevented LC3 II formation and decreased Beclin 1 and p62 expression in response to oxysterol treatment. These metabolic inhibitors were used at the relevant concentrations reported in the literature and also tested as not being cytotoxic in the experimental model adopted [19]. Accumulating research now interest in modulating autophagy for cancer therapy. Of note, MEK/ERK and PI3K/Akt pathways are activated in several cancers and contribute to cancer progression [53], [54]. Therefore, targeting these pathways to regulate autophagy could be a promising tool for cancer therapy to maintain autophagic response at sufficient levels.