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In the present study we investigate the role of c-FLIP in autophagosome biogenesis. c-FLIP is an apoptosis modulator [1] and plays a complex role in cellular homeostasis [2]. In the last few years a cross-talk between autophagy and apoptosis has been highlighted [3], but this complex mechanism still remains partially unknown. In c-FLIP-/- MEFs (mouse embryonic fibroblasts) compared to WT MEFs we analysed two well-known autophagy markers, LC3 and p62, under different autophagy-inducing stimuli (torin 1, starvation and tunicamycin). We found a strong reduction of the autophagic flux in c-FLIP-/- MEFs. We then studied the activation state of specific markers at each stage of the autophagic process and c-FLIP was found to participate in the nucleation stage and to bind key factors in the autophagosomes nucleation. Then we analysed the autophagic flux at increasing times and doses after treatment with autophagic inducers. A positive correlation was observed between death increase and autophagic flux induction in WT MEFs. Cell death was partially reversed by combining drug treatments with autophagy inhibitors. On the contrary, autophagy inhibition did not affect the basal low-level death of c-FLIP-/- MEFs. Further experiments are currently ongoing to better characterize the involved mechanisms and we can conclude that c-FLIP protein absence reduces autophagy-dependent cell death. [ABSTRACT FROM AUTHOR]

Extra virgin olive oil, containing high levels of monounsaturated fatty acids such as oleic acid, has been shown to exert several protective effects on the liver, reducing hepatic steatosis, fibrogenesis and lipid peroxidation. Remarkably, oleic acid is known for its anti-cancer effects in different tumors through cell death induction [1] and autophagy modulation [2]. The aim of the present study is to investigate the effects of oleic acid on hepatocellular carcinoma. More in detail, we are investigating the effects of oleic acid treatment in two hepatocarcinoma cell lines, namely Huh7.5 and Hep3B, by evaluating lipid accumulation (through oil red staining), cell death index (through annexin and propidium iodide assay), expression of endoplasmic reticulum stress- and lipid synthesis- markers as well as analyzing autophagic flux (by Western blot assays). We found that the storage levels of neutral lipids in Huh7.5 are different than in Hep3B cell lines. This is true both under basal conditions and upon oleic acid treatment. Conversely, cell death index is increased in both cell lines upon high oleic acid concentration (300 mM). Unexpectedly, such increased cell death inversely correlates with the endoplasmic reticulum stress marker BIP, since it displays a mild reduction after high oleic acid treatment in both cell lines (300 mM). Interestingly, oleic acid treatment dose-dependently (50 to 150 mM) leads to a reduction of peroxisome proliferator-activated receptor (PPAR)-g expression in both cell lines, thus suggesting a reduction of de novo lipid synthesis at low-dose oleic acid treatments. Furthermore, reduced autophagic flux upon high oleic acid treatment (300 mM) occurs in both cell lines, as demonstrated by LC3 lipidation in the presence of bafilomycin. We are currently investigating if autophagy modulation after oleic acid treatment in these cell lines depends on phospho-glycogen synthase kinase-3 (P-GSK-3) and phospho AMP-activated protein kinase (P-AMPK). We therefore hypothesize that autophagy may have a crucial role in controlling cell death after high oleic acid treatment in the two investigated hepatocarcinoma cell lines, unrevealing new insights on the mechanisms underlying autophagy in hepatocarcinoma. [ABSTRACT FROM AUTHOR]