Identification of novel cholesterol-derived anticancer agents : Application to metabolic therapy of glioblastoma

Le cholestérol est une molécule aux multiples facettes remplissant des fonctions essentielles pour l’organisme. Néanmoins, son excès induit des effets néfastes qui peuvent impliquer soit directement le cholestérol, soit ses dérivés, principalement les oxystérols, qui résultent de l'oxydation du cholestérol. Les connaissances actuelles laissent supposer l’implication du cholestérol et des oxystérols dans la cancérogenèse. Les oxystérols, dont la concentration intracellulaire est corrélée à celle du cholestérol, exercent un rôle actif dans la signalisation cellulaire et l’homéostasie du cholestérol. La dérégulation du métabolisme du cholestérol est impliquée dans plusieurs cancers dont le glioblastome, qui est caractérisé par une biosynthèse régulée à la hausse du cholestérol contribuant à la prolifération et à la survie des cellules tumorales. En raison de leurs caractéristiques physico-chimiques et biologiques certains oxystérols ont un potentiel antitumoral. L’objectif de la Thèse a consisté à étudier les propriétés antitumorales de dérivés du cholestérol (acides biliaires hybrides, oxystérols) et d’analogues végétaux du cholestérol, sur des cellules de glioblastome (lignée de glioblastome de rat C6). Nous avons mis en évidence que les acides biliaires hybrides et les oxystérols varient considérablement dans leur capacité à induire la mort des cellules C6. Dans tous les cas, les molécules cytotoxiques identifiées, dont la diosgénine (phytostérol), induisent un stress oxydant et des dysfonctions mitochondriales et activent l’autophagie. Le 7β-hydroxycholestérol utilisé comme oxystérol cytotoxique de référence induit une mort cellulaire associée à un stress oxydant et à de l’autophagie. Les acides biliaires hybrides toxiques induisent une surproduction d’espèces réactives de l’oxygène, une dépolarisation des mitochondries et activent l’autophagie qui est caractérisée par des modulations d’expression de mTOR, Beclin-1, Atg12, du complexe Atg12-Atg5 et par une augmentation du rapport LC3II / LC3I. Parmi les oxystérols cytotoxiques, le 5β,6β-epoxycholestérol (5,6β-EC) est le plus puissant. Il active fortement l'autophagie, induit un stress oxydant, des dysfonctions mitochondriales et lysosomales et une augmentation de perméabilité de la membrane plasmique. L’absence de cellules avec des noyaux condensés et/ou fragmentés, l’absence de dégradation de PARP et de caspase-3 clivée, sont en faveur d’une mort non apoptotique. Une diminution des ARNm de LXRβ a été observée. L’activité cytotoxique du 5,6β-EC se caractérise par un blocage des cellules en phases (S / G2-M) du cycle cellulaire. Nos résultats montrent que des molécules issues du cholestérol, acides biliaires hybrides et oxystérols, pourraient ainsi être de nouveaux agents anticancéreux. En raison de leur implication potentielle dans le métabolisme du cholestérol, qui est dérégulé dans le glioblastome, ces molécules pourraient être utilisées en vue d’une thérapie métabolique antitumorale du glioblastome.Cholesterol is a multifunctional molecule that performs essential functions for the body. However, its excess induces harmful effects which can involve either cholesterol directly or its derivatives, mainly oxysterols, which result from the oxidation of cholesterol. Current knowledge suggests the involvement of cholesterol and oxysterols in carcinogenesis. Oxysterols, which have intracellular concentration correlated with that of cholesterol, play an active role in cell signaling and cholesterol homeostasis. The deregulation of cholesterol metabolism is implicated in several cancers including glioblastoma, which is characterized by an upregulation of cholesterol biosynthesis contributing to the proliferation and survival of tumor cells. Due to their physicochemical and biological characteristics, some oxysterols have antitumor potential. The objective of this work was to study the antitumor properties of cholesterol derivatives (hybrid bile acids, oxysterols) and phytosterols, on glioblastoma cells (C6 rat glioblastoma cells). We have shown that hybrid bile acids and oxysterols vary considerably in their ability to induce the death of C6 cells. In all cases, the cytotoxic molecules identified, including diosgenin, which is a phytosterol, induce oxidative stress and mitochondrial dysfunctions and activate autophagy. 7β-hydroxycholesterol, used as cytotoxic reference, induces cell death, associated with oxidative stress and autophagy. Toxic hybrid bile acids induce an overproduction of reactive oxygen species and depolarization of mitochondria. They also activate an autophagy characterized by modulations of the expression of mTOR, Beclin-1, Atg12 and Atg12-Atg5 complex in addition to an increase in LC3II / LC3I ratio. Among the cytotoxic oxysterols, 5β, 6β-epoxycholesterol (5,6β-EC) is the most potent. Thus, 5,6β-EC strongly activates autophagy, induces oxidative stress, mitochondrial and lysosomal dysfunctions, and increases the permeability of plasma membrane. No cells with condensed and/or fragmented nuclei, no PARP degradation and no cleaved-caspase-3, which are apoptotic criteria, were observed. A decrease in LXRβ mRNA was identified. The cytotoxic activity of 5,6β-EC is also characterized by a blockage in the (S /G2-M) phases of the cell cycle. Our results show that molecules deriving from cholesterol, hybrid bile acids and oxysterols, could constitute new anticancer agents. Due to their potential involvement in the metabolism of cholesterol, which is deregulated in glioblastoma, these molecules could be used in the antitumor metabolic therapy of glioblastoma

Localisation of oxysterols at the sub-cellular level and in biological fluids

Oxysterols are oxidized derivatives of cholesterol that are formed enzymatically or via reactive oxygen species or both. Cholesterol or oxysterols ingested as food are absorbed and packed into lipoproteins that are taken up by hepatic cells. Within hepatic cells, excess cholesterol is metabolised to form bile acids. The endoplasmic reticulum acts as the main organelle in the bile acid synthesis pathway. Metabolised sterols originating from this pathway are distributed within other organelles and in the cell membrane. The alterations to membrane oxysterol:sterol ratio affects the integrity of the cell membrane. The presence of oxysterols changes membrane fluidity and receptor orientation. It is well documented that hydroxylase enzymes located in mitochondria facilitate oxysterol production via an acidic pathway. More recently, the presence of oxysterols was also reported in lysosomes. Peroxisomal deficiencies favour intracellular oxysterols accumulation. Despite the low abundance of oxysterols compared to cholesterol, the biological actions of oxysterols are numerous and important. Oxysterol levels are implicated in the pathogenesis of multiple diseases ranging from chronic inflammatory diseases (atherosclerosis, Alzheimer’s disease and bowel disease), cancer and numerous neurodegenerative diseases. In this article, we review the distribution of oxysterols in sub-cellular organelles and in biological fluids

Sources of 7-ketocholesterol, metabolism and inactivation strategies: food and biomedical applications

Graphical abstract Abstract 7-Ketocholesterol (or 7-oxocholesterol) is an oxysterol essentially formed by cholesterol autoxidation. It is often found at enhanced levels in the body fluids and/or target tissues of patients with age-related diseases (cardiovascular, neuronal, and ocular diseases) as well as in subjects concerned with civilization diseases (type 2 diabetes, bowel diseases, and metabolic syndrome). The involvement of increased 7-ketocholesterol levels in the pathophysiology of these diseases is widely suspected. Indeed, 7-ketocholesterol at elevated concentrations is a powerful inducer of oxidative stress, inflammation, and cellular degeneration which are common features of all these diseases. It is important to better know the origin of 7-ketocholesterol (diet, incidence of environmental factors, and endogenous formation (autoxidation and enzymatic synthesis)) and its inactivation mechanisms which include esterification, sulfation, oxidation, and reduction. This knowledge will make it possible to act at different levels to regulate 7-ketocholesterol level and counteract its toxicity in order to limit the incidence of diseases associated with this oxysterol. These different points as well as food and biomedical applications are addressed in this review