title: Role of D-Limonene in autophagy induced by bergamot essential oil in SH-SY5Y neuroblastoma cells.
abstract: Bergamot (Citrus bergamia, Risso et Poiteau) essential oil (BEO) is a well characterized, widely used plant extract. BEO exerts anxiolytic, analgesic and neuroprotective activities in rodents through mechanisms that are only partly known and need to be further investigated. To gain more insight into the biological effects of this essential oil, we tested the ability of BEO (0.005-0.03%) to modulate autophagic pathways in human SH-SY5Y neuroblastoma cells. BEO-treated cells show increased LC3II levels and appearance of dot-like formations of endogenous LC3 protein that colocalize with the lysosome marker LAMP-1. Autophagic flux assay using bafilomycin A1 and degradation of the specific autophagy substrate p62 confirmed that the observed increase of LC3II levels in BEO-exposed cells is due to autophagy induction rather than to a decreased autophagosomal turnover. Induction of autophagy is an early and not cell-line specific response to BEO. Beside basal autophagy, BEO also enhanced autophagy triggered by serum starvation and rapamycin indicating that the underlying mechanism is mTOR independent. Accordingly, BEO did not affect the phosphorylation of ULK1 (Ser757) and p70(S6K) (Thr389), two downstream targets of mTOR. Furthermore, induction of autophagy by BEO is beclin-1 independent, occurs in a concentration-dependent manner and is unrelated to the ability of BEO to induce cell death. In order to identify the active constituents responsible for these effects, the two most abundant monoterpenes found in the essential oil, d-limonene (125-750 µM) and linalyl acetate (62.5-375 µM), were individually tested at concentrations comparable to those found in 0.005-0.03% BEO. The same features of stimulated autophagy elicited by BEO were reproduced by D-limonene, which rapidly increases LC3II and reduces p62 levels in a concentration-dependent manner. Linalyl acetate was ineffective in replicating BEO effects; however, it greatly enhanced LC3 lipidation triggered by D-limonene.
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical and Translational Pharmacology, University of Calabria, Rende, Cosenza, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Russo
authors.affiliation: Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.
authors.forename: Maria Gilda Valentina
authors.initials: MG
authors.lastname: Cassiano
authors.affiliation: Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Ciociaro
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical and Translational Pharmacology, University of Calabria, Rende, Cosenza, Italy.
authors.forename: Annagrazia
authors.initials: A
authors.lastname: Adornetto
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical and Translational Pharmacology, University of Calabria, Rende, Cosenza, Italy.
authors.forename: Giuseppe Pasquale
authors.initials: GP
authors.lastname: Varano
authors.affiliation: Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Chiappini
authors.affiliation: Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Berliocchi
authors.affiliation: Headache Science Centre, National Neurological Institute "C. Mondino," and Department of Brain and Behavioural Sciences, University of Pavia, Pavia, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Tassorelli
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical and Translational Pharmacology, University of Calabria, Rende, Cosenza, Italy.
authors.forename: Giacinto
authors.initials: G
authors.lastname: Bagetta
authors.affiliation: Department of Health Sciences, University "Magna Graecia" of Catanzaro, Catanzaro, Italy.
authors.forename: Maria Tiziana
authors.initials: MT
authors.lastname: Corasaniti
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D053138:Cyclohexenes / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D000077222:Limonene; D008247:Lysosomes / Q000378:metabolism; D061986:MCF-7 Cells; D008561:Membrane Fusion / Q000187:drug effects; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D039821:Monoterpenes / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D009822:Oils, Volatile / Q000494:pharmacology*; D010588:Phagosomes / Q000378:metabolism; D010938:Plant Oils / Q000494:pharmacology*; D013729:Terpenes / Q000494:pharmacology*
chemical_list: D053138:Cyclohexenes; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D039821:Monoterpenes; D009822:Oils, Volatile; D010938:Plant Oils; D013729:Terpenes; C068336:bergamot oil; C074463:linalyl acetate; D000077222:Limonene