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The role of autophagy is known to be highly complex and context-dependent, leading to both cancer suppression and progression in several tumors including melanoma, breast and prostate cancer. In the present review, recent advances in an understanding of the involvement of autophagy in prostate cancer treatment are described. The regulatory effects of androgens on prostate cancer cell autophagy are particularly discussed in order to highlight the effects of autophagy modulation during androgen deprivation. A critical evaluation of the studies examined in the present review suggests the attractive possibility of autophagy inhibition combined with hormonal therapy as a promising approach for prostate cancer treatment. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiatingmelanosphere-derived cells. Increased lipid uptakewas found inmelanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor- γ (PPAR-γ ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology. [ABSTRACT FROM AUTHOR]

Cellular-Flice-like inhibitory protein (c-FLIP) is an apoptosis modulator known to inhibit the extrinsic apoptotic pathway thus blocking Caspase-8 processing in the Death Inducing Signalling Complex (DISC). We previously demonstrated that c-FLIP localizes at the endoplasmic reticulum (ER) and that c-FLIP-deficient mouse embryonic fibroblasts (MEFs) display an enlarged ER morphology. In the present study, we have addressed the consequences of c-FLIP ablation in the ER stress response by investigating the effects of pharmacologically-induced ER stress in Wild Type (WT) and c-FLIP −/− MEFs. Surprisingly, c-FLIP −/− MEFs were found to be strikingly more resistant than WT MEFs to ER stress-mediated apoptosis. Analysis of Unfolded Protein Response (UPR) pathways revealed that Pancreatic ER Kinase (PERK) and Inositol-Requiring Enzyme 1 (IRE1) branch signalling is compromised in c-FLIP −/− cells when compared with WT cells. We found that c-FLIP modulates the PERK pathway by interfering with the activity of the serine threonine kinase AKT. Indeed, c-FLIP −/− MEFs display higher levels of active AKT than WT MEFs upon ER stress, while treatment with a specific AKT inhibitor of c-FLIP −/− MEFs subjected to ER stress restores the PERK but not the IRE1 pathway. Importantly, the AKT inhibitor or dominant negative AKT transfection sensitizes c-FLIP −/− cells to ER stress-induced cell death while the expression of a constitutively active AKT reduces WT cells sensitivity to ER stress-induced death. Thus, our results demonstrate that c-FLIP modulation of AKT activity is crucial in controlling PERK signalling and sensitivity to ER stress, and highlight c-FLIP as a novel molecular player in PERK and IRE1-mediated ER stress response. [ABSTRACT FROM AUTHOR]

Different stressful conditions such as hypoxia, nutrient deprivation, pH changes, or reduced vascularization, potentially able to act as growth-limiting factors for tumor cells, activate the unfolded protein response (UPR). UPR is therefore involved in tumor growth and adaptation to severe environments and is generally cytoprotective in cancer. The present review describes the molecular mechanisms underlying UPR and able to promote survival and proliferation in cancer. The critical role of UPR activation in tumor growth promotion is discussed in detail for a few paradigmatic tumors such as prostate cancer and melanoma. [ABSTRACT FROM AUTHOR]

c-Flip proteins are well-known apoptosis modulators. They generally contribute to tissue homeostasis maintenance by inhibiting death-receptor-mediated cell death. In the present manuscript, we show that c-Flip knock-out (KO) mouse embryonic fibroblasts (MEFs) kept in culture under starvation conditions gradually modify their phenotype and accumulate vacuoles, becoming progressively larger according to the duration of starvation. Large vacuoles are present in KO MEFs though not in WT MEFs, and are Oil Red-O positive, which indicates that they represent lipid droplets. Western blot experiments reveal that, unlike WT MEFs, KO MEFs express high levels of the lipogenic transcription factor PPAR-γ. Lipid droplet accumulation was found to be associated with endoplasmic reticulum (ER) stress activation and autophagic modulation valuated by means of BIP increase, LC3 lipidation and AMP-activated protein kinase (AMPK) phosphorylation, and p62 accumulation. Interestingly, XBP-1, an ER stress-induced lipogenic transcription factor, was found to preferentially localize in the nucleus rather than in the cytoplasm of KO MEFs. These data demonstrate that, upon starvation, c-Flip affects lipid accumulation, ER stress and autophagy, thereby pointing to an important role of c-Flip in the adaptive response and ER stress response programs under both normal and pathological conditions. [ABSTRACT FROM AUTHOR]

Angiopoietins are vascular factors essential for blood vessel assembly and correct organization and maturation. This study describes a novel calcium-dependent machinery activated through Angiopoietin-1/2-Tie receptor system in HUVECs monolayer. Both cytokines were found to elicit intracellular calcium mobilization. Targeting intracellular Ca2+ signaling, antagonizing IP3 with 2-APB or cADPR with 8Br-cADPR, was found to modulate in vitro angiogenic responses to Angiopoietins in a specific way. 2-APB and 8Br-cADPR impaired the phosphorylation of AKT and FAK induced by Ang-1 and Ang-2. On the other hand, phosphorylation of ERK1/2 and p38, as well as cell proliferation, was not affected by either inhibitor. The ability of ECs to migrate following Angs stimulation, evaluated by “scratch assay,” was reduced by either 2-APB or 8Br-cADPR following Ang-2 stimulation and only slightly affected by 2-APB in cells stimulated with Ang-1. These results identify a novel calcium-dependent machinery involved in the complex interplay regulating angiogenic processes showing that IP3- and cADPR-induced Ca2+ release specifically regulates distinct Angs-mediated angiogenic steps. [ABSTRACT FROM AUTHOR]

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the upregulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression. [ABSTRACT FROM AUTHOR]

Necroptosis is a form of programmed necrosis whose molecular players are partially shared with apoptotic cell death. Here we summarize what is known aboutmolecular signalling of necroptosis, particularly focusing on fine tuning of FLIP and IAP proteins in the apoptosis/necroptosis balance. We also emphasize necroptosis involvement in physiological and pathological conditions, particularly in the regulation of immune homeostasis. [ABSTRACT FROM AUTHOR]

Toll-like receptor 3 (TLR3) is a key effector of the innate immune system against viruses. Activation of TLR3 exerts an antitumoral effect through a mechanism of action still poorly understood. Here we show that TLR3 activation by polyinosinic:polycytidylic acid induces up-regulation of microRNA-29b, -29c, -148b, and -152 in tumor-derived cell lines and primary tumors. In turn, these microRNAs induce reexpression of epigenetically silenced genes by targeting DNA methyltransferases. In DU145 and TRAMP-C1 prostate and MDA-MB-231 breast cancer cells, we demonstrated that polyinosinic:polycytidylic acid-mediated activation of TLR3 induces microRNAs targeting DNA methyltransferases, leading to demethylation and reexpression of the oncosuppressor retinoic acid receptor beta (RARβ). As a result, cancer cells become sensitive to retinoic acid and undergo apoptosis both in vitro and in vivo. This study provides evidence of an antitumoral mechanism of action upon TLR3 activation and the biological rationale for a combined TLR3 agonist/retinoic acid treatment of prostate and breast cancer. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) recognize microbial/viral--derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C) induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-inducible factor 1 (HIF-1) regulates several cellular processes, including apoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumormachinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific I.3 isoform of HIF-1α and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF). Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1α. However, the transfection of I.3 isoformof hif-1α in LNCaP cells allows poly(I:C)--induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1α expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists. [ABSTRACT FROM AUTHOR]

Background: Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis. Methodology/Principal Findings: By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2- induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90b is the main player in this phenomenon. Conclusions/Significance: We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90. [ABSTRACT FROM AUTHOR]

Objective: To study the effect of c-FLIP overexpression in testicular germ cells. Design: A novel transgenic mouse model overexpressing the apoptotic modulator c-FLIP in the testis was generated. Setting: Animal facility and university research laboratory. Animal(s): Transgenic mice overexpressing the long isoform of c-FLIP (c-FLIPL) under the transcriptional control of a 400 bp long regulatory region of the Stra8 promoter. Intervention(s): Spermatozoa motility and testis histological, immunohistochemical, and Western blot analyses were carried out in transgenic and control derived specimens. Main Outcome Measure(s): Testis morphology, sperm motility, and germ cell apoptosis were assayed. Results: Stra8 promoter was found to activate the ectopic overexpression of c-FLIPL in round and elongated spermatids. As a consequence of such overexpression, a dramatic loss of germ cells was observed, resulting in testicular atrophy associated with reduced sperm motility. Conclusion(s): The data show that c-FLIPL forced expression in haploid male germ cells has detrimental effects on spermatogenesis and sperm quality and reveal a possible mechanism underlying the onset of testicular atrophy. [Copyright &y& Elsevier]

We have investigated the role of NAADP-mediated Ca2+ mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca2+ release which was blocked by inhibitory concentrations of NAADP, by impairing Ca2+ uptake in acidic stores with bafilomycin, and by thapsigargin. Ca2+ release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca2+ signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca2+ responses were antagonized by disruption of lipid rafts/caveolae with methyl-β-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca2+ signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling. J. Cell. Physiol. 216: 396–404, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

When chronically stimulated with agonists of contraction, smooth muscle cells (SMCs) undergo cell hypertrophy, a process defined as increase in size and potentiation of the contractile phenotype in the absence of proliferation. Hypertrophic response has long been associated to a number of pathologies of the cardiovascular and respiratory systems. We have investigated the phenotypic and functional response of SMCs to long-term treatment with endothelin. Our model was primary cultures of peritubular smooth muscle cells (PSMC) a testicular cell type target of locally produced endothelin and characterized by an unusual phenotypic stability when cultured in simple medium in complete absence of serum. We report the following responses of PSMC to 4-day exposure to ET-1: (i) increased protein synthesis without induction of cell proliferation; (ii) increase in cell size (evaluated by means of flow cytometry) and increased expression of SM-α-actin, desmin, caldesmon and calponin, markers of the contractile phenotype. In experiments of selective stimulation of either ETA or ETB receptor subtypes, both proved to be involved in inducing the observed hypertrophic responses. The hypertrophic cells exhibit the ultrastructural features of differentiated SMCs and are capable of calcium mediated contractile response when acutely stimulated with ET-1 specifically through ETA and/or ETB receptors, as evaluated by calcium imaging and scanning electron microscopy. These observations demonstrate that engagement of ET receptors is capable of inducing potentiation of the contractile phenotype and functional hypertrophy of PSMC. J. Cell. Physiol. 212: 264–273, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis represents a fundamental process during fetal/post-natal testis development. Therefore pro- and anti-apoptotic proteins are essential to regulate testis physiology. c-FlipL is a known inhibitor of caspase 8/10 activity; in this study its perinatal expression in mouse male germ cells was investigated. In testis sections and seminiferous tubule whole mount c-FlipL was found to be expressed in undifferentiated spermatogonia and to co-localize with germ stem cells markers. In vivo investigations in the vitamin-A deficient mouse, lacking differentiated germ cells, confirmed c-FlipL expression in undifferentiated spermatogonia. Further analyses showed Fas expression but no significant caspase 8/10 activity when c-FlipL was highly expressed. Altogether these data suggest that c-Flip may control the survival rate of undifferentiated spermatogonia. [Copyright &y& Elsevier]

Peritubular smooth muscle cells (PSMC) from rat testis in primary serum-free cultures unexpectedly undergo contraction and subsequent cell hypertrophy in response to the growth factor PDGF-BB, remaining stationary. The present study investigates the transduction pathways involved in the observed paradoxical upregulation of the differentiated phenotype and induction of hypertrophy in PSMC. PI3K, ERK, JNK, and p38 kinases, known to mediate PDGF-BB signaling in the canonic dedifferentiative and proliferative response of smooth muscle cells (SMC) were rapidly activated by PDGF-BB but only p38 remained activated after 2-day stimulation. Immunofluorescence and immunoblotting experiments showed that in 4-day treatment: (i) continuous inhibition of PI3K, of ERK, of JNK, failed to inhibit either cell enlargement and formation of prominent α-SM actin containing stress fibers or the typical increase in α-SM actin; (ii) when stimulated in the presence of the p38 inhibitor SB203580 both responses were significantly inhibited and cytofluorimetric analysis of cell size showed a remarkable reduction of the hypertrophic response. PDGF-BB was also found to activate the small GTPase RhoA and inhibition of Rho-dependent kinase ROCK by Y27632 counteracted the effects of PDGF-BB similarly to SB203580. Both the transcription factor ATF2 and the nucleosomal kinase MSK1, downstream targets of p38, were activated by PDGF-BB, but p38 inhibitor SB203580 inhibited only the phosphorylation of MSK1 which appeared unaffected by ROCK inhibitor Y27632. In concluding, p38 and the Rho-ROCK system were found to play prominent, probably independent roles in the upregulation of PSMC differentiated phenotype and induction of hypertrophy by PDGF-BB. J. Cell. Physiol. 207: 123–131, 2006. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to investigate the expression and role of c-Flip long isoform (c-FlipL), a known anti-apoptotic protein. No data are currently available on c-FlipL in male gonad before puberty; therefore, this study was carried out in prepuberal mouse testis. We investigated pachytene spermatocytes and spermatogonia by immunostaining of testis sections and found a strong and specific expression of c-FlipL in pachytene spermatocytes, while spermatogonia expressed very low levels of c-FlipL. This finding inversely correlated with the caspases activity, which was higher in spermatogonia as compared to pachytene spermatocytes. Other experiments carried out in an organ-culture model revealed that Fas-induced apoptosis was higher in spermatogonia as compared to pachytene spermatocytes. These data suggest that c-FlipL may play a role as an anti-apoptotic molecule in the prepuberal mouse testis and open new perspectives in the comprehension of the mechanisms controlling germ cells apoptosis. [Copyright &y& Elsevier]

Abstract: The contractile activity of peritubular tissue is responsible for the propulsion of spermatozoa along the lumen of seminiferous tubules toward the hilum of the testis. This function is performed by specialized contractile cells [peritubular smooth muscle cells (PSMC)] in response to the locally produced agonist, endothelin (ET). Here, we review current information on the complex ET-mediated control of peritubular contractility. In addition, we report new data demonstrating that the relaxant peptide adrenomedullin is produced by Sertoli cells and interferes with ET-mediated contraction of PSMC. Given the relevance of the seminiferous tubule sperm output for male fertility, the detailed definition of the mechanisms controlling peritubular contractility could contribute in different ways to novel therapeutic opportunities and provide potential targets for contraceptive strategies. [Copyright &y& Elsevier]

Analyzes the expression of fatty acid synthase ligand (FasL) in different testicular cell types, during testis development as well as various stages of spermatogenesis. Presence of FasL in mature spermatozoa indicating its involvement in the self-defense mechanism of gametes against antisperm-activated lymphocytes; Protein localization of FasL.

The article discusses c-Flip expression and function in fetal mouse genocytes. c-Flip is expressed in fetal male mouse gonads and its expression is developmentally regulated. Fas-dependent apoptosis in fetal testes is dependent on c-Flip levels. Initiator caspase-10 is expressed in fetal male mouse gonad and its activity is developmentally regulated.

Investigates the signal transduction mechanisms involved in Fas induction. Pretreatment of Sertoli cells with specific inhibitors of ERKs and P38 at all concentrations tested; Regulation of Fas expression to avoid indiscriminate cell death; Significance of understanding the molecular mechanisms activated by cytokines.

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]