Laura Berliocchi1, Carlotta Chiappini1, Annagrazia Adornetto2, Debora Gentile1, Silvia Cerri3, Rossella Russo2, Giacinto Bagetta4, Maria Tiziana Corasaniti5. 1. Department of Health Sciences, University "Magna Graecia" of Catanzaro, Campus Universitario "Salvatore Venuta", V Livello Edificio delle Bioscienze, Viale Europa, 88100 Catanzaro, Italy. 2. Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical and Translational Pharmacology, University of Calabria, 87036 Rende (Cosenza), Italy. 3. Center for Research in Neurodegenerative Diseases, C. Mondino National Neurological Institute, 27100 Pavia, Italy. 4. Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical and Translational Pharmacology, University of Calabria, 87036 Rende (Cosenza), Italy. Electronic address: g.bagetta@unical.it. 5. Department of Health Sciences, University "Magna Graecia" of Catanzaro, Campus Universitario "Salvatore Venuta", V Livello Edificio delle Bioscienze, Viale Europa, 88100 Catanzaro, Italy. Electronic address: mtcorasa@unicz.it.
Abstract
BACKGROUND: d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. PURPOSE: To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. METHODS: Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. RESULTS: LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and stimulates ROS generation, yet none of these events is implicated in lipidation of LC3, which was only partly reduced by chelation of intracellular calcium. The early LC3 lipidation induced by d-limonene is associated with inhibition of clonogenic capacity which is reverted by the autophagy inhibitor chloroquine. CONCLUSIONS: d-Limonene rapidly stimulates the autophagic flux in cultured cancer cells, which could be usefully exploited for therapeutic purposes.
BACKGROUND:d-Limonene is a natural monoterpene abundant in Citrus essential oils. It is endowed with several biological activities, including inhibition of carcinogenesis and promotion of tumour regression. Recently, d-limonene has been shown to modulate autophagic markers in vitro at concentrations found in vivo, in clinical pharmacokinetic studies. Autophagy is an intracellular catabolic process serving as both an adaptive metabolic response and a quality control mechanism. Because autophagy defects have been linked to a wide range of human pathologies, including neurodegeneration and cancer, there is a need for new pharmacological tools to control deregulated autophagy. PURPOSE: To better understand the effects of d-limonene on autophagy, to identify the molecular mechanisms through which this monoterpene rapidly triggers LC3 lipidation and to evaluate the role for autophagy in long-term effects of d-limonene. METHODS:Human SH-SY5Y neuroblastoma, HepG2 hepatocellular carcinoma and MCF7 breast cancer cells were used. Endogenous LC3-II levels were evaluated by western blotting. Autophagic flux assay was performed using bafilomycin A1 and chloroquine. Intracellular distribution of LC3 protein was studied by confocal microscopy analysis of LC3B-GFP transduced cells. Expression of lysosomal-membrane protein LAMP-1 was assessed by immunofluorescence analysis. Phosphorylated levels of downstream substrates of mTOR kinase (p70S6 kinase, 4E-BP1, and ULK1) and ERK were analyzed by western blotting. Production of reactive oxygen species (ROS) was assessed by live confocal microscopy of cells loaded with CellROX® Green Reagent. Clonogenic assay was used to evaluate the ability of treated cells to proliferate and form colonies. RESULTS:LC3 lipidation promoted by d-limonene correlates with autophagosome formation and stimulation of basal autophagy. LC3 lipidation does not rely on inhibition of mTOR kinase, which instead appears to be transiently activated. In addition, d-limonene rapidly activates ERK and stimulates ROS generation, yet none of these events is implicated in lipidation of LC3, which was only partly reduced by chelation of intracellular calcium. The early LC3 lipidation induced by d-limonene is associated with inhibition of clonogenic capacity which is reverted by the autophagy inhibitor chloroquine. CONCLUSIONS:d-Limonene rapidly stimulates the autophagic flux in cultured cancer cells, which could be usefully exploited for therapeutic purposes.
Authors: Damiana Scuteri; Maria Roberta Garreffa; Stefania Esposito; Giacinto Bagetta; Maria Diana Naturale; Maria Tiziana Corasaniti Journal: Neural Regen Res Date: 2018-09 Impact factor: 5.135
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