The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor compound, has not yet been fully elucidated. Here, we show that human cancer cells have markedly lower levels of sphingomyelin (SM) than nontumor (MRC-5) cells. In this context, 2OHOA treatment strongly augments SM mass (4.6-fold), restoring the levels found in MRC-5 cells, while a loss of phosphatidylethanolamine and phosphatidylcholine is observed (57 and 30%, respectively). The increased SM mass was due to a rapid and highly specific activation of SM synthases (SMS). This effect appeared to be specific against cancer cells as it did not affect nontumor MRC-5 cells. Therefore, low SM levels are associated with the tumorigenic transformation that produces cancer cells. SM accumulation occurred at the plasma membrane and caused an increase in membrane global order and lipid raft packing in model membranes. These modifications would account for the observed alteration by 2OHOA in the localization of proteins involved in cell apoptosis (Fas receptor) or differentiation (Ras). Importantly, SMS inhibition by D609 diminished 2OHOA effect on cell cycle. Therefore, we propose that the regulation of SMS activity in tumor cells is a critical upstream event in 2OHOA antitumor mechanism, which also explains its specificity for cancer cells, its potency, and the lack of undesired side effects. Finally, the specific activation of SMS explains the ability of this compound to trigger cell cycle arrest, cell differentiation, and autophagy or apoptosis in cancer cells.anticancer | membrane-lipid therapy | lung cancer | membrane lipids T he potent antitumor compound 2-hydroxyoleic acid (2OHOA) (Minerval®) acts against cancer by inducing cell cycle arrest (1-3), followed by apoptosis in human leukemia cells (4) or differentiation and autophagy in the case of human glioma cells. Despite the potency of 2OHOA against cancer, it is a safe nontoxic compound with IC 50 values in nontumor cells 30-to 150-fold greater than in tumor cells (4). The high efficacy and low toxicity of this fatty acid produce a wide therapeutic window that can only be the consequence of a highly specific mechanism of action, the molecular bases of which have, in part, been elucidated here.The 2OHOA compound was designed rationally to reproduce the antitumor effect of anthracyclines via interactions with the plasma membrane and the ensuing modifications in cell signaling (5), without unspecific interactions with other cell targets. It is known that 2OHOA binds to membranes and modifies the biophysical properties of the lipid bilayer, the first target encountered by this synthetic lipid (6). Nevertheless, the regulatory effects of 2OHOA on the composition of cancer cell membranes have yet to be described. In fact, 2OHOA induces changes in the localization and activity of membrane proteins involved in cancer cell proliferation, differentiation and survival, such as the Fas receptor (4), PKC (3), as well as cyclins, cyclin-dependent kinases (CDKs), caspases, E2F-1 and dihydrofolate reductase...

Despite recent advances in the development of new cancer therapies, the treatment options for glioma remain limited, and the survival rate of patients has changed little over the past three decades. Here, we show that 2-hydroxyoleic acid (2OHOA) induces differentiation and autophagy of human glioma cells. Compared to the current reference drug for this condition, temozolomide (TMZ), 2OHOA combated glioma more efficiently and, unlike TMZ, tumor relapse was not observed following 2OHOA treatment. The novel mechanism of action of 2OHOA is associated with important changes in membrane-lipid composition, primarily a recovery of sphingomyelin (SM) levels, which is markedly low in glioma cells before treatment. Parallel to membrane-lipid regulation, treatment with 2OHOA induced a dramatic translocation of Ras from the membrane to the cytoplasm, which inhibited the MAP kinase pathway, reduced activity of the PI3K/Akt pathway, and downregulated Cyclin D-CDK4/6 proteins followed by hypophosphorylation of the retinoblastoma protein (RB). These regulatory effects were associated with induction of glioma cell differentiation into mature glial cells followed by autophagic cell death. Given its high efficacy, low toxicity, ease of oral administration, and good distribution to the brain, 2OHOA constitutes a new and potentially valuable therapeutic tool for glioma patients.fatty acids | sphingomyelin synthase | cancer drug target | glioma biomarker C ancer cells of undifferentiated phenotype (e.g., glioma) have a poor prognosis and limited treatment options. Primary brain tumors, of which glioma is the most common, are generally associated with very high rates of mortality (ca. 90%), being the median survival of patients about 1 y (1, 2). Chemotherapy provides only modest benefits to radiotherapy and surgery being the alkylating agent temozolomide (TMZ) the reference drug; however, tumor relapse is usually observed, and TMZ only increases the patients' life expectancy about 2.5 m (from 12.1 to 14.6 m: ref.3). The present study was designed to investigate the efficacy of 2OHOA against glioma and its molecular mechanisms of action. 2OHOA exhibited a greater efficacy than TMZ in the treatment of glioma, and there was no relapse after long-term treatment with 2OHOA. This efficacy and lack of toxicity at therapeutic doses has been acknowledged recently by the European Medicines Agency (EMA) to designate 2OHOA orphan drug for the treatment of glioma. In previous studies, we showed that this compound induces cell cycle arrest of lung cancer cells (4-6). Here, we showed that 2OHOA reversed the altered lipid profile of glioma cells and how this modification regulated cell signaling to induce autophagy specifically in glioma but not normal cells.Moreover, in the present study we demonstrated that the changes induced by 2OHOA were specific to cancer cells with no significant effects observed in normal cells and no adverse effects in treated animals, features not shared by most anticancer drugs. The efficacy of this compound in the absence ...

Alzheimer's disease (AD) is a neurodegenerative pathology with relevant unmet therapeutic needs. Both natural aging and AD have been associated with a significant decline in the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), and accordingly, administration of DHA has been proposed as a possible treatment for this pathology. However, recent clinical trials in mild-to-moderately affected patients have been inconclusive regarding the real efficacy of DHA in halting this disease. Here, we show that the novel hydroxyl-derivative of DHA (2-hydroxydocosahexaenoic acid - OHDHA) has a strong therapeutic potential to treat AD. We demonstrate that OHDHA administration increases DHA levels in the brain of a transgenic mouse model of AD (5xFAD), as well as those of phosphatidylethanolamine (PE) species that carry long polyunsaturated fatty acids (PUFAs). In 5xFAD mice, administration of OHDHA induced lipid modifications that were paralleled with a reduction in amyloid-β (Αβ) accumulation and full recovery of cognitive scores. OHDHA administration also reduced Aβ levels in cellular models of AD, in association with alterations in the subcellular distribution of secretases and reduced Aβ-induced tau protein phosphorylation as well. Furthermore, OHDHA enhanced the survival of neuron-like differentiated cells exposed to different insults, such as oligomeric Aβ and NMDA-mediated neurotoxicity. These results were supported by model membrane studies in which incorporation of OHDHA into lipid-raft-like vesicles was shown to reduce the binding affinity of oligomeric and fibrillar Aβ to membranes. Finally, the OHDHA concentrations used here did not produce relevant toxicity in zebrafish embryos in vivo. In conclusion, we demonstrate the pleitropic effects of OHDHA that might prove beneficial to treat AD, which suggests that an upstream event, probably the modulation of the membrane lipid composition and structure, influences cellular homeostasis reversing the neurodegenerative process. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Background2-Hydroxyoleic acid is a synthetic fatty acid with potent anti-cancer activity which does not induce undesired side effects. However, the molecular and cellular mechanisms by which this compound selectively kills human glioma cancer cells without killing normal cells is not fully understood. The present study was designed to determine the molecular bases underlying the potency against 1321N1, SF-767 and U118 human glioma cell lines growth without affecting non cancer MRC-5 cells.Methodology/Principal FindingsThe cellular levels of endoplasmic reticulum (ER) stress, unfolded protein response (UPR) and autophagy markers were determined by quantitative RT-PCR and immunoblotting on 1321N1, SF-767 and U118 human glioma cells and non-tumor MRC-5 cells incubated in the presence or absence of 2OHOA or the ER stress/autophagy inducer, palmitate. The cellular response to these agents was evaluated by fluorescence microscopy, electron microscopy and flow cytometry. We have observed that 2OHOA treatments induced augments in the expression of important ER stress/UPR markers, such as phosphorylated eIF2α, IRE1α, CHOP, ATF4 and the spliced form of XBP1 in human glioma cells. Concomitantly, 2OHOA led to the arrest of 1321N1 cells in the G2/M phase of the cell cycle, with down-regulation of cyclin B1 and Cdk1/Cdc2 proteins in the three glioma cell lines studied. Finally, 2OHOA induced autophagy in 1321N1, SF-767 and U118 cells, with the appearance of autophagic vesicles and the up-regulation of LC3BI, LC3BII and ATG7 in 1321N1 cells, increases of LC3BI, LC3BII and ATG5 in SF-767 cells and up-regulation of LC3BI and LC3BII in U118 cells. Importantly, 2OHOA failed to induce such changes in non-tumor MRC-5 cells.Conclusion/SignificanceThe present results demonstrate that 2OHOA induces ER stress/UPR and autophagy in human glioma (1321N1, SF-767 and U118 cell lines) but not normal (MRC-5) cells, unraveling the molecular bases underlying the efficacy and lack of toxicity of this compound.