Role of D-Limonene in autophagy induced by bergamot essential oil in SH-SY5Y neuroblastoma cells.

Rossella Russo,Giacinto Bagetta,Maria Tiziana Corasaniti,Laura Berliocchi,Maria Gilda Valentina Cassiano,Antonella Ciociaro,Giuseppe Pasquale Varano,Cristina Tassorelli,Annagrazia Adornetto,Carlotta Chiappini

doi:10.1371/journal.pone.0113682

Rossella Russo, Giacinto Bagetta + Show 8 more

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https://doi.org/10.1371/journal.pone.0113682

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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 p70S6K (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.

Highlights

Autophagy is an intracellular catabolic process by which cytosolic materials are enclosed by a double-membraned structure, forming an autophagosome that is delivered to, and fuses with lysosomes for proteolytic degradation [1]
Our previous data show that a significant percentage of apoptotic and necrotic cell death occurs within 1 h exposure to 0.02% bergamot essential oil (BEO) and this dramatically increases in SH-SY5Y cells incubated for the same time period with 0.03% BEO; no cytotoxic effects are observed following incubation with lower concentrations (0.005–0.01%) of BEO for 1 h and up to 24 h [22,23,24]
To analyze the autophagic flux, the effects of BEO were studied in the presence of the lysosomal inhibitor bafilomycin A1 (BafA1), which blocks autophagosome-lysosome fusion [28], preventing LC3II degradation [29]

Summary

Introduction

Autophagy is an intracellular catabolic process by which cytosolic materials are enclosed by a double-membraned structure, forming an autophagosome that is delivered to, and fuses with lysosomes for proteolytic degradation [1]. This process acts as an adaptive metabolic response by recycling cellular components under conditions of nutrient limitations, and as an intracellular quality control system by promoting turnover of long-lived proteins, removal of damaged organelles, degradation of misfolded and aggregate-prone proteins, elimination of intracellular bacteria [1]. In recent years, converging data have been accumulated linking dysregulated autophagy to diverse pathologies, from cancer to muscle, liver, cardiac, infectious, immune, inflammatory, and neurodegenerative diseases [2]. Defective autophagy has been reported in human tumors with monoallelic deletions of the autophagy gene beclin and in cancer cells in which gain-of-function mutations or somatic amplifications in the oncogenes PI3K and Akt lead to constitutive activation of the autophagy-suppressing PI3K/mTOR pathway [2]

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