BACKGROUND

Glioblastoma multiforme (GBM), also called more simply glioblastoma, is the most common and lethal primary brain tumor that exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, high recurrence rate and apoptosis resistance as well as resistance to traditional therapeutic approaches including radio-chemotherapy (1 ,2). In brain tumors, the challenges of the different therapies available require to pass the blood-brain barrier (BBB) which often contributes to treatment failure (3). GBM has a low survival rate, 12 to 15 months, fewer than 3 to 7% of people surviving longer than five years (4), with current standard therapy which includes surgery, radiation and temozolomide. Only countries with advanced health care systems can provide highly specialized radiotherapy and neuro oncology services (5). Because of the invasive nature of GBM, the entire tumor cannot be removed surgically (6). Despite optimal treatment, GBM usually recurs and without treatment, survival is around three months (7). GBM is the most aggressive diffuse glioma of astrocytic lineage and is considered a grade IV glioma (4), making up 54% of all gliomas and 16% of all primary brain tumors (5). Therapeutic targeting for GBM are very challenging due to its features, and consequently the development of new treatments, such as metabolic anti-cancer therapy, is therefore necessary. As the cholesterol biosynthesis, is implicated in tumor development and cancer progression (8), the cholesterol pathway has emerged as a potentially new target for GBM (9). The metabolic approach aims to suppress the tumorigenicity of GBM through cholesterol pathway leading to cell death using modulators of cholesterol metabolism such as oxysterols which are oxygenated derivatives of cholesterol.

Understanding the role of cholesterol metabolism and transport in GBM cells and the underlying mechanisms of cholesterol-related drug resistance could lead to the development of targeted and more effective therapies for GBM.

The brain cholesterol pool and its metabolism, is distinct from other organs due to the inability of peripheral cholesterol to cross the blood-brain barrier (BBB) (10). In fact, peripheral and central nervous system (CNS) cholesterol metabolism are regulated independently. Indeed, peripherally cholesterol depends on the balance between dietary intake and hepatic synthesis alternatively its degradation, whereas in CNS, cholesterol is synthetized de novo b astrocytes and delivered to neurons to ensure brain physiology (11 ,12). Cholesterol uptake provided by the astrocytes is a crucial step for growth and survival for GBM cells (10). The cholesterol produced and secreted by astrocytes is supplied to the GBM cells by Apo-lipoproteins E (Apo-E). GBM cells rely on astrocytes to supply themselves with cholesterol. Oxysterols and other cholesterol derivatives produced in neurons following cholesterol uptake and metabolism can be physiological agonists for LXRa/p (9). Oxysterols inhibit cholesterol synthesis and enhance its export by activating LXRs (13,14). Activation of LXRs results in its dimerization with retinoid X receptor (RXR), favoring cholesterol efflux through sterol transporters such as ATP-binding cassette subtype-A1 (ABCA1 ), which is the main exporter of cholesterol in the form of Apo-E; and suppressing cholesterol uptake through MYLIP also known as IDOL (Inducible Degrader of the LDL receptor) (10,15,16). The E3 ligase IDOL, is transcriptionally up- regulated by LXR/RXR in response to an increase in intracellular cholesterol (17). IDOL targets the low-density lipoprotein receptor (LDLR) for degradation (16). The LXR-IDOL-LDLR mechanism results in a decrease in cholesterol uptake, thereby regulating the level of intracellular cholesterol (10). In GBM cells, these cholesterol regulatory and surveillance mechanisms occurring in normal glial and nervous cells are disrupted (9,10).

There are several approaches involving cholesterol metabolism known in the GBM field, all of which have the same goal which is the depletion of intracellular cholesterol thus leading to cell death.

The LXR-IDOL-LDLR axis is a common targetable pathway in GBM (18). The LXR agonists, GW3965 hydrochloride and LXR-623 (WAY 252623), which have not a steroid structure (19,20), up-regulate the expression of E3 ubiquitin ligase IDOL which results in reducing LDLR levels. They also up-regulate the expression of cholesterol transporter gene ABCA1 , which then induces substantial apoptosis via activation of the LXRp isoform (10,18). GW3965 and LXR623 have showed anti-GBM activity on U87- MG and GBM39 GBM cells as well as in a xenograft model of intracranial GBM.

As well, phytol and retinol, inhibitors of SREBP-1 synthesis, are able to induce GBM cell death by interfering with fatty acid and cholesterol metabolism (21 ).

With archazolid B, which is also a non-steroid molecule, the expression of LDLR is upregulated, leading to an increase in extracellular cholesterol uptake. This drug hampers the action of V-ATPase due to a proton transport defect. This leads to associated increases in lysosomal pH, thereby preventing cholesterol recycling (22).

Statins may also potentially serve as a new therapeutic approach for combination therapy in GBM (23). The effect of statins may be due to regulation of autophagy by the mevalonate cascade (24,25). The mevalonate pathway modulates autophagy through geranylgeranylation of the small GTPase molecule Rab11 (24). The mevalonate pathway is responsible for the production of the isoprenoids such as geranylgeranyl-pyrophosphate (geranylgeranyl-PP) that play an important role in the prenylation of the superfamily of Ras-like GTPase proteins known as the Rab family (26). Rab GTPases are involved in vesicular trafficking where Rabi 1 and Rab7 are critical components for autophagosome formation and autophagosome-lysosome fusion (27). Thus, inhibition of the mevalonate pathway by statins results in decreased prenylation of Rab11 and Rab7 and subsequent inhibition of autophagy flux (26,27). Therefore the inhibition of the mevalonate pathway followed by autophagy inhibition leads to apoptotic cell death (25,28). SUMMARY OF THE INVENTION

The present invention is directed to the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment of cancer.

The present invention also relates to a pharmaceutical composition comprising the compound 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

Said cancer is selected from the group consisting of: brain cancer, neuroblastoma, hematopoietic cancer, sarcoma, breast cancer, prostate cancer, ovary cancer, carcinoma, lung cancer, kidney cancer and melanoma. Said brain cancer may be a glioma, in particular an astrocytoma and more particularly a glioblastoma.

In a particular embodiment, said use according to the invention is in combination with one or more active molecules.

DETAILED DESCRIPTION

The Inventors have discovered that oxysterols, in particular epoxycholesterols such as 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I), were new effective molecules for the treatment of cancer.

Thus, according to a first aspect, the present invention relates to the compound 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. According to the present disclosure, “pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.

A "pharmaceutically acceptable salt" form of an active ingredient may initially confer a desirable pharmacokinetic property on the active ingredient which were absent in the non-salt form, and may even positively affect the pharmacodynamics of the active ingredient with respect to its therapeutic activity in the body. The phrase “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include acid addition salts, formed with inorganic acids such as sulfuric acid, succinic acid, hydrochloric acid, hydrobromic acid, methanesulfonic acid, p-toluenesulfonic acid, carbonic acid, photonic acid, acetic acid, oxalic acid, tartaric acid, mandelic acid, fumaric acid, maleic acid, lactic acid, citric acid, glutamic acid, saccharic acid, monomethylimbic acid, 5-oxoproline acid, hexane-1 -sulphonic acid, nicotinic acid, 2, 3 or 4-aminobenzoic acid, 2,4,6-trimethylbenzoic acid, lipoic acid, aspartic acid, hydrochloric acid. Other examples are mono- or bis-oxalates salt.

In particular, a pharmaceutically acceptable salt of 5,6p-EC is the 5p, 6p-epoxycholesterol sulfate or the 5p, 6p-epoxycholesterol succinyl.

According to the present invention, the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) may be glycosylated, it means that a sugar can be grafted to the compound. Alternatively, a polyethylene glycol may be grafted to the compound. In particular, such a modification is perfomed by a chemical reaction on the OH in position 3 of 5,6p-EC.

All the embodiments described in the present application for 5,6p-EC also apply a pharmaceutically acceptable salt thereof and to said grafted 5,6p-EC.

According to the present disclosure, the term “treating”, with regard to a subject, refers to improving at least one symptom of the subject's disorder. Treating can be curing, improving, or at least partially ameliorating the disorder.

In a particular embodiment, said cancer is selected from the group consisting of: brain cancer, neuroblastoma, hematopoietic cancer, sarcoma, breast cancer, prostate cancer, ovary cancer, carcinoma, lung cancer, kidney cancer, melanoma.

Thus, in a particular embodiment, the present invention relates to the compound 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer selected from the group consisting of: brain cancer, neuroblastoma, hematopoietic cancer, sarcoma, breast cancer, prostate cancer, ovary cancer, carcinoma, lung cancer, kidney cancer, melanoma. Brain cancers include primary brain tumors which are those that originate in the brain such as glioma (29) or medulloblastoma. Brain is a frequent metastasic site for tumors originating elsewhere in the body. Secondary or metastatic brain tumors arise following the spread of cancer, such as neuroblastoma, from the primary site.

In a particular embodiment, said brain cancer is glioma or medulloblastoma. More particularly, said glioma may selected from the group consisting of: astrocytoma, oligodendrogliomas, oligoastrocytoma and ependymomas. Said astrocytoma may be a glioblastoma which is the more aggressive astrocytoma (30).

Therefore, in a particular embodiment, the present invention relates to the compound 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of a brain cancer, said brain cancer being a glioma such as astrocytoma, oligodendroglioma, oligoastrocytoma and ependymoma, or a medulloblastoma.

In a more particular embodiment, the present invention relates to 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of astrocytoma.

In a more particular embodiment, the present invention relates to 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of glioblastoma.

In a particular embodiment, said hematopoietic cancer is leukemia, lymphoma or myeloma.

Therefore, in a particular embodiment, the present invention relates to the compound 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of a hematopoietic cancer selected among leukemia, lymphoma and myeloma.

In a particular embodiment, the compound 5p, 6p-epoxycholesterol of formula (I) or a pharmaceutically acceptable salt thereof for use as described above, is administered in combination with one or more active molecule. In particular, the at least one active molecule is selected among: lipid-lowering agents, such as hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (also called statins), and/or anticancer agents such as anticancer chemical compounds like temozolomide and/or such as antibodies for example immune checkpoint inhibitors (ICIs) which are monoclonal antibodies directed against the immune checkpoints of the immune system.

It should be noted that lipid-lowering agents may be also called hypolipidemic agents, cholesterol- lowering drugs or antihyperlipidemic agents.

In this case, it can be spoken of a combined preparation or combined product, i.e. the compound or a salt thereof, and the other active molecule are not gathered in a same composition but are separate, and both administrated to the patient in need. The compound or a salt thereof and the other active molecule may then be administered simultaneously, separate or sequentially.

Thus, the present invention relates to the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, as described above, in particular in the treatment of the glioblastoma, in combination with at least one active molecule, said at least one active molecule is in particular a lipid-lowering agent such as statin, and/or an anti-cancer agent such as temozolomide or an antibody a monoclonal antibody directed against the immune checkpoints.

In a more particular embodiment, the compound 5,6p-EC or a salt thereof is administered in combination with temozolomide and/or a hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor.

Therefore, in a particular embodiment, the present invention relates to the compound 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer, particularly the treatment of a glioblastoma, in combination with temozolomide and/or a hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor.

In a particular embodiment, said compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, may be conjugated to a compound that facilitates its transport across the blood-brain barrier (BBB). As used herein, a compound which facilitates transport across the BBB is one which, when conjugated to 5,6p-EC, facilitates the amount of peptide delivered to the brain as compared with non-conjugated 5,6p-EC. The compound can induce transport across the BBB by any mechanism, including receptor-mediated transport, and diffusion.

Compounds which facilitate transport across the BBB include transferrin receptor binding antibodies; certain lipoidal forms of dihydropyridine; carrier peptides, such as cationized albumin or Met- enkephalin; cationized antibodies; fatty acids such as docosahexaenoic acid (DHA) and C8 to C24 fatty acids with 0 to 6 double bonds, glyceryl lipids, polyarginine (e.g., RR, RRR, RRRR) and polylysine (e.g., KK, KKK, KKKK). Unbranched, naturally occurring fatty acids embraced by the invention include C8:0 (caprylic acid), C10:0 (capric acid), C12:0 (lauric acid), C14:0 (myristic acid), C16:0 (palmitic acid), C16:1 -7 (palmitoleic acid), C16:2, C18:0 (stearic acid), C18:1 -9 (oleic acid), C18:1 -7 (vaccenic), C18:2-6 (linoleic acid), C18:3-3 (alpha-linolenic acid), C18:3-5 (eleostearic), C18:3-6 (alpha-linolenic acid), C18:4-3, C20:1 -9 (gondoic acid), C20:2-6, C20:3-6 (dihomo-gamma- linolenic acid), C20:4-3, C20:4-6 (arachidonic acid), C20:5-3 (eicosapentaenoic acid), C22:1 (docosenoic acid), C22:4-6 (docosatetraenoic acid), C22:5-6 (docosapentaenoic acid), C22:5-3 (docosapentaenoic), C22:6-3 (docosahexaenoic acid) and C24:1 -9 (nervonic). Other BBB carrier molecules and methods for conjugating such carriers to 5,6p-EC will be known to one of ordinary skill in the art.

In the event that 5,6p-EC exhibits reduced activity in a conjugated form, the covalent bond between 5,6p-EC and the BBB transport-mediating compound can be selected to be sufficiently labile (e.g., to enzymatic cleavage by an enzyme present in the brain) so that it is cleaved following transport of 5,6p- EC across the BBB, thereby releasing free 5,6p-EC to the brain. Biologically labile covalent linkages, e.g., imino bonds, and "active" esters can be used to form prodrugs. A second aspect of the invention relates to a pharmaceutical composition comprising the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

In a particular embodiment, said cancer is selected from the group consisting of: brain cancer, neuroblastoma, hematopoietic cancer, sarcoma, breast cancer, prostate cancer, ovary cancer, carcinoma, lung cancer, kidney cancer, melanoma.

Therefore, in a particular embodiment, the present invention relates to a pharmaceutical composition comprising the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer selected from the group consisting of: brain cancer, neuroblastoma, hematopoietic cancer, sarcoma, breast cancer, prostate cancer, ovary cancer, carcinoma, lung cancer, kidney cancer, melanoma.

In a particular embodiment, said brain cancer is glioma or medulloblastoma. More particularly, said glioma may selected from the group consisting of: astrocytoma, oligodendrogliomas, oligoastrocytoma and ependymomas. In particular, said astrocytoma may be a glioblastoma.

Therefore, in a particular embodiment, the present invention relates to a pharmaceutical composition comprising the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) for use in the treatment of a brain cancer, said brain cancer being a glioma such as astrocytoma, oligodendroglioma, oligoastrocytoma and ependymoma, or a medulloblastoma.

In a more particular embodiment, the present invention relates to a pharmaceutical composition comprising the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of astrocytoma.

In a more particular embodiment, the present invention relates to a pharmaceutical composition comprising the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of glioblastoma.

In a particular embodiment, said hematopoietic cancer is leukemia, lymphoma or myeloma.

Thus, in a particular embodiment, the present invention relates to a pharmaceutical composition comprising the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, for use in the treatment of a hematopoietic cancer selected among leukemia, lymphoma and myeloma.

In a particular embodiment, said pharmaceutical composition further comprises one or more active molecule. In particular, the at least one active molecule is selected among: lipid-lowering agents, such as hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (also called statins), and/or anticancer agents such as anticancer chemical compounds like temozolomide and/or such as antibodies for example immune checkpoint inhibitors (ICIs) which are monoclonal antibodies directed against the immune checkpoints of the immune system.

Thus, the present invention relates to a pharmaceutical composition comprising the compound 5,6p- EC of formula (I) or a pharmaceutical salt thereof for use as described above, said pharmaceutical composition further comprising at least one active molecule, said at least one active molecule is in particular a lipid-lowering agent such as statin, and/or an anti-cancer agent such as temozolomide or a monoclonal antibody directed against the immune checkpoints.

In a more particular embodiment, said pharmaceutical composition further comprises temozolomide and/or a hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor.

Therefore, in a particular embodiment, the present invention relates to a pharmaceutical composition comprising the compound 5,6p-EC of formula (I) or a pharmaceutical salt thereof for use in the treatment of cancer, particularly the treatment of a glioblastoma, wherein said pharmaceutical composition further comprises temozolomide and/or a hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor.

In particular, said pharmaceutical composition comprises a pharmaceutically acceptable carrier.

The term “carrier” refers to a compound that is useful in the preparation of a pharmaceutical composition, generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for human pharmaceutical uses. It encompasses carriers, excipients, and diluents and means a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body.

Useful pharmaceutical carriers for the preparation of the compositions hereof, can be solids or liquids. The carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., nigella seed oil, mineral oil, and the like. Water, saline, aqueous dextrose, and glycols are representative liquid carriers, particularly (when isotonic with the blood) for injectable solutions. For example, formulations for intravenous administration comprise sterile liquid solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in an appropriated vehicle to produce a liquid solution and rendering the solution sterile.

Suitable pharmaceutical carriers include starch, cellulose, talc, glucose, lactose, tragacanth, gelatin, pectin, dextrin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, methylcellulose, sodium carboxymethylcellulose, dried skim milk, glycerol, propylene glycol, ethanol, and the like. The pharmaceutical composition may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, colorants, flavors, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like. The pharmaceutical composition of the invention may have a form of solid (e. g. include powders, tablets, coated tablets, dragees, pills, capsules, cachets, suppositories, and dispersible granules), or liquid (e. g. solutions, emulsions, syrups, elixirs, or suspensions).

The compound 5p, 6p-epoxycholesterol of formula (I), a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, as described above, may be administered, for example, ocularly (ocular route), orally (e.g., buccal cavity), sublingually, parenterally (e.g., intramuscularly, intravenously, or subcutaneously), rectally (e.g., by suppositories), transdermally (e.g., skin electroporation) or by nasal route.

In case of brain cancer treatment, such as glioblastoma treatment, the compound 5p, 6p- epoxycholesterol of formula (I), a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, as described above, may be administered by any of the routes listed above.

The application of MRI-guided focused ultrasound and the administration of bradykinin, will weaken or even open the blood-brain barrier (BBB), allowing the drugs to diffuse through it.

To reach the brain tumor without constraining the passage of the BBB, an intra-tumoral administration of compound 5p, 6p-epoxycholesterol of formula (I), a pharmaceutically acceptable salt thereof or the pharmaceutical composition, as described above, may be carried out. The compound is then injected into the tumor with a syringe needle or using a catheter.

Administration by the nasal route is another way to reach brain tumors without the constraint of the passage of the BBB.

The compound 5p, 6p-epoxycholesterol of formula (I), a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, as described above, may be in a suitable vehicle. They may be microencapsulated, such as in a liposome. They may be delivered by nanoparticles. In this embodiment, they may be nanoencapsulated, that is to say inside the nanoparticles, or the compound 5,6 p-EC may be linked to the surface of the nanoparticles, for example when using metal nanoparticles, such as gold (Au) or iron oxide nanoparticles. Then, in some embodiments the microparticles and the nanoparticles may be functionalized. Moreover, if nanoparticles are magnetic or superparamagnetic, they may be guided in a magnetic field. There are several examples of microparticles and nanoparticles, functionalized or not, which may be used in cancer treatment, particularly in glioblastoma treatment (29).

The present invention also concerns a use of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, or a use of a pharmaceutical composition comprising 5p, 6p- epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof as described above, in the manufacture of a medicament for treating cancer. In a particular embodiment, the present invention concerns the use of 5p, 6p-epoxycholesterol (5,6p- EC) of formula (I) or a pharmaceutically acceptable salt thereof, or a use of a pharmaceutical composition comprising 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof as described above, in the manufacture of a medicament for treating a cancer selected from the group consisting of: brain cancer, neuroblastoma, hematopoietic cancer, sarcoma, breast cancer, prostate cancer, ovary cancer, carcinoma, lung cancer, kidney cancer, melanoma.

In a particular embodiment, the present invention relates to the use of 5p, 6p-epoxycholesterol (5,6p- EC) of formula (I) or a pharmaceutically acceptable salt thereof, or a use of a pharmaceutical composition comprising 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof as described above, in the manufacture of a medicament for treating a brain cancer, said brain cancer being a glioma such as astrocytoma, oligodendroglioma, oligoastrocytoma and ependymoma, or a medulloblastoma.

In a more particular embodiment, the present invention relates to the use of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof, or a use of a pharmaceutical composition comprising 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof as described above, in the manufacture of a medicament for treating astrocytoma, more particularly for treating glioblastoma.

In a particular embodiment, the present invention concerns the use of 5p, 6p-epoxycholesterol (5,6p- EC) of formula (I) or a pharmaceutically acceptable salt thereof, or a use of a pharmaceutical composition comprising 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof as described above, in the manufacture of a medicament for treating a hematopoietic cancer selected among leukemia, lymphoma and myeloma.

In a particular embodiment, said use of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof above described, is in combination with one or more active molecule, said active molecule is in particular is selected among: lipid-lowering agents, such as hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (also called statins), and/or anticancer agents such as anticancer chemical compounds like temozolomide and/or such as antibodies for example immune checkpoint inhibitors (ICIs) which are monoclonal antibodies directed against the immune checkpoints of the immune system.

The present invention is further directed to a method for the treatment of cancer, comprising a step of administering to a patient in need thereof a therapeutically effective amount of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof, or of a pharmaceutical composition, as described above.

The term "therapeutically effective amount" as used herein means an amount required to reduce symptoms of the disease in an individual. The dose will be adjusted to the individual requirements in each particular case. That dosage can vary within wide limits depending upon numerous factors such as the severity of the disease to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved.

In a particular embodiment, the present invention relates to a method for the treatment of cancer, comprising a step of administering to a patient in need thereof a therapeutically effective amount of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof, or of a pharmaceutical composition, as described above, said cancer being selected from the group consisting of: brain cancer, neuroblastoma, hematopoietic cancer, sarcoma, breast cancer, prostate cancer, ovary cancer, carcinoma, lung cancer, kidney cancer, melanoma.

In a particular embodiment, the present invention relates to a method for the treatment of brain cancer, comprising a step of administering to a patient in need thereof a therapeutically effective amount of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof, or of a pharmaceutical composition, as described above, said brain cancer being a glioma such as astrocytoma, oligodendroglioma, oligoastrocytoma and ependymoma, or a medulloblastoma.

In a more particular embodiment, the present invention concerns a method for the treatment of astrocytoma, more particularly glioblastoma, comprising a step of administering to a patient in need thereof a therapeutically effective amount of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof, or of a pharmaceutical composition, as described above.

In a particular embodiment, the present invention relates to a method for the treatment of hematopoietic cancer, comprising a step of administering to a patient in need thereof a therapeutically effective amount of 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof, or of a pharmaceutical composition, as described above, said hematopoietic cancer being selected from the group consisting of: leukemia, lymphoma and myeloma.

In a particular embodiment, said 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a pharmaceutically acceptable salt thereof is administered simultaneously, separately or sequentially in combination with one or more active molecule, said active molecule is in particular selected among: lipid-lowering agents, such as hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (also called statins), and/or anticancer agents such as anticancer chemical compounds like temozolomide and/or such as antibodies for example immune checkpoint inhibitors (ICIs) which are monoclonal antibodies directed against the immune checkpoints of the immune system.

All the embodiments relative to the therapeutically use of a pharmaceutical composition comprising the compound 5p, 6p-epoxycholesterol (5,6p-EC) of formula (I) or a salt thereof, described in the present application, also apply in the case where said compound 5,6p-EC or a salt thereof is conjugated to a compound that facilitates its transport across the blood-brain barrier (BBB), as previously defined. FIGURES

Figure 1 : Heatmap representation of the anti-tumor activities of oxysterols and hybrid bile acids used on C6 glioblastoma cells. The activities are visualized in grey scale: the weakest are in light grey, the strongest in black.

Figure 2: Effects of 5,6-EC (5,6p-EC and 5,6a-EC) and TMZ on C6 cell viability.

Figure 3: Impact of 5,6- EC (5,6p-EC and 5,6a-EC) and TMZ on cell morphology studied by phase contrast microscopy.

Figure 4: Evaluation of the effects of 5,6-EC (5,6p-EC and 5,6a-EC) and TMZ on ROS production in C6 cells.

Figure 5: Sterol profile after treatment of C6 cells with 5,6p-EC

EXAMPLE 1 : STUDY OF THE ANTI-TUMORAL ACTIVITY

MATERIAL & METHODS

1. Tested molecules

Cytotoxicity activities have been studied for the following natural sterols and synthetic sterols:

1 ) Natural sterols: 7p-hydroxycholesterol (7p-OHC), 22(R)-hydroxycholesterol (22R-OHC), 24(S)- hydroxycholesterol (24(S)-OHC), 5a,6a-Epoxycholesterol (5,6a-EC)), 5p,6p-Epoxycholesterol (5,6p- EC).

2) Synthetic sterols: 22(R)-ISO-hydroxycholesterol (22R-ISO-OHC), ((23- (4-methylfuran-2, 5-dione) -

3a-hydroxy-24-nor-5(3-cholane) (LITHO 1 a), 23- (4-methylfuran-2, 5-dione) - 3a, 7a-dihydroxy-24-nor- 5(3-cholane) (CHENO 1 b), 23-(4-methyl-1 H-pyrrole-2,5-dione)-3a-hydroxy-24-nor-5(3-cholane) (LITOMAL 7a), 23-(4-methyl-1 H-pyrrole-2,5-dione)-3a,7a, 12a-trihydroxy-24-nor-5(3-cholane)

(COLMAL 7f) and ethanol maleimide derivatives of litocholic and chenocholic acid (LITOMET, CHENOMET).

These sterols were synthesized by Prof. M. Samadi (chemistry department, LCP A2MC, University of Lorraine, Metz, France). The purity of these sterols was determined to be 100% by gaseous phase chromatography and mass spectrometry.

2. Cell culture and treatments

C6 rat glioblastoma cells have been chosen as a screening model to assess cytotoxicity as they are known for their extreme resistance to cell death as well as for their use both in vitro and in vivo. Indeed, C6 cells can also be implanted in rats by forming subcutaneous or intracerebral gliomas. C6 rat glioblastoma cells were seeded at 240,000 cells per well in 6-well microplates containing 2 mL of culture medium (Dulbecco’s modified Eagle medium (DMEM) (Lonza) supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS) (Pan Biotech) and 1% antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin) (Pan Biotech)). C6 cells were incubated at 37°C in a humidified atmosphere containing 5% CO2, and passaged twice a week. At each passage, cells were trypsinized with a (0.05% trypsin - 0.02% EDTA) solution (Pan Biotech). After 24 h of culture, C6 cells were further treated for 24 h with various concentrations of sterols: 2.5, 5, 10, 20, 40 and 80 pg/mL (range of concentrations: 5.5 to 180 pM).

3. Phase contrast microscopy

Cell morphology (adherent and non-adherent cells) and cell density was observed after 24 h of culture on untreated and treated cells under an inverted-phase contrast microscope (Axiovert 40CFL, Zeiss) at x400 magnification. For each condition of culture, 9 fields in the center of the well were observed. Digitized images were obtained with a video camera (Axiocam ICm1 , Zeiss)

4. Cell counting (trypan blue)

After trypsinization, C6 cells were centrifuged and resuspended in culture medium. The total number of viable cells was determined in the presence of trypan blue after 24 h of treatment in a range of concentrations from 5.5 mM to 180 mM under an inverted phase contrast microscope. This range of concentrations was chosen in order to calculate with accuracy the half maximal inhibitory concentration (IC50) corresponding to the concentration required to reduce by 50% the number of viable cells.

5. MTT assay

The MTT assay was used to evaluate the effects of treatments on cell proliferation and/or mitochondrial activity. The MTT assay was carried out on C6 cells plated in 6-well flat bottom culture plates after 24 h of culture on untreated and treated cells. MTT salt (Sigma- Aldrich) is reduced to formazan in the metabolically active cells by the mitochondrial enzyme succinate dehydrogenase. The plates were read at 570 nm with a microplate reader -Tecan Sunrise (Tecan).

6. Measurement of Cell Viability with the Fluorescein Diacetate (FDA) assay

Cell viability was determined by staining with fluorescein diacetate (FDA) by measuring esterase’s activity. The FDA assay was carried out on C6 cells plated in 6-well flat bottom culture plates after 24 h of culture on untreated and treated cells. At the end of the treatment, cells were incubated for 10 min at 37 °C with 15 pg/mL fluorescein diacetate (FDA, Sigma-Aldrich), rinsed twice with phosphate buffer saline (PBS) and lysed with a Tris/HCI solution containing 1% sodium dodecyl sulfate (SDS) (Sigma- Aldrich). Fluorescence was measured with excitation at 485 nm and emission at 528 nm using a plate reader-Tecan Sunrise (Tecan). 7. Cell clonogenic survival assay

Cell clonogenic survival assay or colony formation assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony following insult with chemicals or radiation. At the end of treatment in 6-well plates, cells were trypsinized, counted on a Malassez hemocytometer in the presence of trypan blue (v/v), and 1 ,000 living cells were cultured in a 100 mm Petri dish in 10 mL of culture medium for 6 days. Cells permeable to propidium iodide (PI: 1 pg/mL) corresponding either to dead cells or to cells with altered plasma membranes were determined by flow cytometry at the beginning of the culture (Day 0). After 6 days of culture, the cells were stained with crystal violet to visualize cell colonies; the medium was changed twice a week.

8. Morphological characterization of apoptotic cells: evaluation of nuclear morphology with Hoechst 33342

Nuclear morphology of control (untreated cells), and treated cells was characterized by fluorescence microscopy after staining with Hoechst 33342 (Sigma-Aldrich) (2 pg/mL). Cell deposits of about 40,000 cells were applied to glass slides by cytocentrifugation (5 min, 1 ,500 rpm) with a cytospin 2 (Shandon), mounted in Dako fluorescent mounting medium (Dako) and stored in the dark at 4°C until observation. The morphological aspect of the cell nuclei was determined with an Axioskop fluorescent microscope (Zeiss). For each sample, 300 nuclei were examined.

9. Measurement of transmembrane mitochondrial potential with DiOCo(3)

Variations in the transmembrane mitochondrial potential (A^Pm) were measured with 3,3'dihexyloxacarbocyanine iodide (DiOCe(3) (Thermo Fisher Scientific)). Adherent and non-adherent cells were pooled and stained with DiOCe(3) (40 nM). Carbonyl cyanide mcholorophenylhydrazone (CCCP; Sigma-Aldrich), a potent mitochondrial oxidative phosphorylation uncoupler, was used as positive control to induce loss of AMJm; to this end, CCCP was prepared at 10 mM in DMSO (Sigma- Aldrich); CCCP (50 pM) was added on the cells 10 min before staining with DiOCe(3). Mitochondrial depolarization is indicated by decreased green fluorescence collected through a 520/20 nm band pass filter on a Galaxy flow cytometer (Partec). Ten thousand cells were acquired; data were analysed with Flomax (Partec) or FlowJo (Tree Star Inc.) softwares.

10. Lysosomal staining with acridine orange

Acridine orange (AO) is a weak base which accumulates in its charged form within lysosomes of living cells because of the low lysosomal pH, and which produces a red fluorescence when excited by a blue light. During prolonged exposure to cytotoxic agents, including oxysterols, the red fluorescence of AO decreases markedly. The shift in AO fluorescence from granular red to diffuse green reflects leakage and redistribution of AO from the lysosomes, indicating impairment of the lysosomal membranes or the ability of the lysosomes to maintain low pH. According to these considerations, AO is widely used to perform lysosomal integrity measurements and the percentage of AO-negative cells (which do not emit a red fluorescence) determined by flow or image cytometry permits quantification of the percentage of cells with destabilized lysosomes. In the present investigation, a 1 mg/ml stock solution of AO (Sigma) was prepared by dissolving the dye in distilled water. After staining with AO (1 volume of AO at 10 pg/ml mixed with 1 volume of cell suspension, incubation for 15 min at 37°C), cells were washed twice in culture medium, resuspended in culture medium, and immediately analyzed by flow cytometry.

11. Flow cytometric evaluation of plasma membrane permeability and cell death by staining with propidium iodide

Cell mortality was measured with propidium iodide (PI). PI, a hydrophilic probe, is an intercalator of nucleic acids. When excited by a blue light (488 nm), PI produces a red/orange fluorescence. PI enters in the cells with damaged, permeabilized plasma membranes which are considered as dead cells. Ten thousand cells were acquired for each sample, and the data were analyzed with FlowJo (Tree Star Inc., Ashland, OR, USA) software. All assays were performed with at least in three independent experiments.

12. Cell cycle analysis

Flow cytometric analysis of the cell cycle was realized on adherent and non-adherent cells collected by trypsinization. Cells were pooled, washed with PBS, and 1 -2 x 106 cells were stained with propidium iodide (PI). Briefly, cells were resuspended in 80% cold ethanol, washed in PBS, and resuspended in PBS containing 80 pg/mL PI and 200 pg/mL RNase A. After incubation (1 h, 37°C), 1 - 2 mL of PBS were added, and flow cytometric analyses were performed on a Galaxy flow cytometer (Partec). The parameters, forward scatter (FSC) and side scatter (SSC), were used to exclude cell debris; FSC and SSC were measured on a logarithmic scale. In addition, after gating on FSC-SSC, doublet cells were excluded by an additional gating on FL2-w (width) versus FL2-a (area); all events inside this gate correspond to single cells and were used to determine the DNA content (FL2-a). FL2a allows to measure the fluorescence of PI, which was collected using a 590/10 nm bandpass filter, and measured on a linear scale. 10,000 cells were acquired and data were analysed with Flomax (Partec) or FlowJo (Tree Star Inc.) softwares.

13. Polyacrylamide gel electrophoresis and Western blotting

Cells were lysed in a RIPA buffer and the protein concentration was measured in the supernatant using the Bicinchoninic Acid Solution (Sigma-Aldrich). Seventy micrograms of proteins were diluted in loading buffer (125 mM Tris-HCI, pH 6.8, 10 % p-mercaptoethanol, 4.6 % SDS, 20 % glycerol, and 0.003 % bromophenol blue), separated on a polyacrylamide SDS-containing gel, and transferred onto a nitrocellulose membrane (Thermo-Scientific). After blocking nonspecific binding sites for 1 h with 5 % nonfat milk in TBST (10 mM Tris-HCI, 150 mM NaCI, 0.1 % Tween 20, pH 8), the membrane was incubated overnight with the primary antibody diluted in TBST with 1 -5 % milk. The antibodies directed against caspase-3 (# 9662 Cell Signaling (Ozyme)) and LC3-I/II (# L8918 (Sigma-Aldrich)), which are rabbit antibodies, were used at 1/1000 final dilution. An antibody raised against p-actin (mouse monoclonal antibody; Ref: A2228 Sigma-Aldrich) was used at 1/10,000 final dilution. The membrane was washed with TBST and incubated (1 h, room temperature) with horseradish peroxidase- conjugated goat anti-mouse (Cell Signaling, # 7074) or anti-rabbit antibody (Santa-Cruz Biotechnology, # sc-2005) diluted at 1 /5,000. The membrane was washed with TBST and revealed using an enhanced chemiluminescence detection kit (Supersignal West Femto Maximum Sensitivity Substrate, Thermo-Scientific) and Chemidoc XRS+ (Bio-Rad). The ratio LC3-II/LC3-I was calculated with Image Lab software (Bio-Rad).

14. Statistical analysis

Statistical analyses and determination of IC50 values were done using XLSTAT software (Microsoft). Data were expressed as mean ± SD; data were considered statistically different (t-student test) at a P- value of 0.05 or less. Heatmap was made with GraphPad Prism 9.

RESULTS

Comparison of the natural and synthetic sterols above-listed has been performed though different tests on rat C6 glioblastoma cells. The tests allow to study the toxicity of the molecules at the level of different cell targets. It has thus been evaluated the effects on cell morphology by phase contrast microscopy, on cell viability by the MTT test, on esterase activity by the FDA test, on cell survival by the clonogenicity test, on mitochondria by measuring the mitochondrial transmembrane potential by staining with 3,3'-dihexyloxacarbocyanine iodide (DiOCe(3)), on the plasma membrane also indicating cell mortality by propidium iodide (PI) staining, on lysosomes by acridine orange (AO) staining, on the cell cycle by detection of cells in phase (G2+M) after PI staining, on autophagy by quantification of LC- 3II and LC-3I protein expression by Western blot (LC-3I l/LC-31 ratio). PI, DiOCe(3) and AO staining are measured by flow cytometry.

An objective classification of the tested sterols has been established considering their toxicity, which has allowed to define that 5p, 6p-epoxycholesterol (5,6p-EC) is the most cytotoxic and the most powerful inducer of autophagy.

5,6p-EC is capable of triggering a non-apoptotic type of cell death on C6 cells, involving the mitochondrial pathway and associated with several features of autophagy. 5,6p-EC is able to strongly modify the cell cycle by blocking the G2M phase. In vitro data obtained on C6 glioblastoma cells reveal antagonistic effects of the enantiomeric 5,6-epoxide. For the 5a, 6a-epoxycholesterol (5,6a- EC), It has been observed, at 45 pM, effects of stimulation of tumor cell proliferation while the 5p, 6p- epoxycholesterol has marked cytotoxic effects at 45 pM suggesting the possibility of using it as a new anti-cancer agent or anti-cancer adjuvant, in particular on glioblastoma cells.

Results are gathered in the table 1 below. Table 1 : Comparison of natural and synthetic sterols

The concentration of 45 pM was chosen for all molecules to perform the different tests. This concentration was chosen based on the ICsos of the MTT and FDA tests, considering in addition the analysis of cell morphology by phase contrast microscopy.

For clonogenicity, 1 means no effect (colony formation as in the control), 2 weak effect, 3 medium effect and 4 strong effect (no colony formation).

Based on these tests (Table 1), a multidimensional and multivariate heatmap was made. The heatmap obtained (Figure 1) allows a comparative study of the cytotoxicity of the studied sterols, including the 5p, 6p-epoxycholesterol which trigger a non-apoptotic mode of cell death with characteristics of autophagy leading an increase of the ratio LC3-II I LC3-I on C6 rat glioblastoma cells.

The C6 cell line is one of the commonest experimental models used in neuro-oncology in order to study GBM and provides a good simulation in overall the high growth rate, the increased vascularization, and the extremely infiltrative character (30). C6 cells are also characterized by their extreme resistance to cell death and for their use both in vitro and in vivo. They have been used to study several biological features of brain tumors, such as tumor growth, tumor invasion and migration, angiogenesis, growth factor production and regulation, and blood-brain barrier disruption (31 ,32). EXAMPLE 2: VIABILITY STUDY - COMPARISON WITH TMZ

The impact of 5,6p-EC and TMZ on viability using FDA test has been studied. TMZ (Temozolomide) is a reference molecule in the field of glioblastoma chemotherapy treatment.

Current treatment of glioblastoma consists of surgical resection of the tumor, followed by 30 weeks of radiotherapy and the use of an adjuvant chemotherapeutic drug, TMZ. This molecule is an alkylating agent that has the ability to cross the BBB and produces cytotoxicity by DNA alkylation (37). TMZ is converted in the bloodstream to an active metabolite: 3-methyl-(triazen-1 -yl) imidazole-4-carboxamide, which gives a methyl group to certain DNA bases (mainly guanine), resulting in a mismatch of O ⁶ - methylguanine with thymidine during DNA replication; this leads to G 2 / M phase arrest of the cell cycle and subsequent cell death (38). However, the therapeutic efficacy of alkylating agents in cancer is limited due to the presence of the repair enzyme methylguanine-O ⁶ -methyltransferase (MGMT), which repairs DNA by directly removing the alkyl group from the genome of cells exposed to alkylating agents (39). Furthermore, as TMZ is an alkylating agent, its activity is not specific and the side effects are significant.

Cell viability was determined by staining with fluorescein diacetate (FDA) by measuring esterase’s activity. The FDA assay was carried out on C6 cells plated in 6-well flat bottom culture plates after 24 h of culture on untreated and treated cells. At the end of the treatment, cells were incubated for 10 min at 37 °C with 15 pg/mL fluorescein diacetate (FDA, Sigma-Aldrich), rinsed twice with phosphate buffer saline (PBS) and lysed with a Tris/HCI solution containing 1% sodium dodecyl sulfate (SDS) (Sigma- Aldrich). Fluorescence was measured with excitation at 485 nm and emission at 528 nm using a plate reader-Tecan Sunrise (Tecan).

The cytotoxic effects of 5,6p-EC on C6 cells were compared to those of temozolomide (TMZ; alkylating agent) which is the reference molecule for current glioblastoma therapy. Under treatment with 5,6p-EC (45 pM), and TMZ (3 mM) for 24 h, marked effects on cell adhesion and growth were observed particularly with 5,6p-EC (Figure 3). The concentrations of 45 pM and 3 mM correspond to the respective IC50 of 5,6p-EC and TMZ. It should be noted that at 45 pM, 5,6p-EC induces cell death while 5,6a-EC and TMZ have no effect at the same concentration. The cytotoxic effect of TMZ on C6 cells is only observed at 3mM, which is a concentration 67 times higher (Figure 2).

The cell death induced by 5,6p-EC is attenuated by a-tocopherol which has no effect on TMZ (Figure 3). By phase contrast microscopy, it has been also observed that dimethyl fumarate (DMF) but especially its major metabolite, monomethyl fumarate (MMF), strongly attenuated the toxicity of 5,6p- EC without having any effect on TMZ, which reinforces the hypothesis of an involvement of oxidative stress in 5,6p-EC-induced cell death.

EXAMPLE 3: ROS PRODUCTION - COMPARISON WITH TMZ

The impact of 5,6p-EC and TMZ on reactive oxygen species (ROS) production has been studied.

To clarify the part taken by oxidative stress in cell death, the overproduction of reactive oxygen species (ROS), superoxide anion (O2-) and hydrogen peroxide (H2O2), induced by 5,6p-EC and TMZ, have been quantified. ROS overproduction was assessed in C6 cells treated for 24 h with 5,6p-EC (45pM), 5,6a-EC (45pM), as well as TMZ (1 .5 and 3 mM). Overproduction of superoxide anion (02*-) and hydrogen peroxide (H2O2) were detected with dihydroethidium (DHE). DHE (1.6 mM) was prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich), and used at 2 pM. After 15 min at 37 °C, the fluorescent signals of DHE stained cells were collected through a 590/20 nm and a 520/20 nm band pass filter on a logarithmic scale on a Galaxy flow cytometer (Partec); 10,000 cells were acquired; data were analyzed with Flomax (Partec) or FlowJo (Tree Star Inc.) softwares.

5,6p-EC activates the overproduction of ROS while 5,6a-EC and TMZ have no effect (Figure 4). These results show that 5,6p-EC-induced cell death is associated with overproduction of ROS, whereas TMZ-induced cell death is not, suggesting that oxidative stress may be one of the key events leading to 5,6p-EC-induced cell death.

EXAMPLE 4: CHARACTERIZATION AND QUATIFICATION OF STEROLS

The characterization and quantification of sterols by gas chromatography coupled with mass spectrometry (GC/MS) has been carried out.

Sterol analysis (7a-hydroxycholesterol, 7p-hydroxycholesterol, 7-ketocholesterol, 5a, 6a- epoxycholesterol, 5p,6p-epoxycholesterol and cholestane-3p, 5a,6p-triol) was realized by GC-MS on a Clarus 600D (Perkin Elmer, USA). The GC was equipped with an Elite column (30 m x 0.32 mm id x 0.25 mm film; Perkin Elmer, USA) and injection was performed in splitless mode using helium (1 mL/min) as carrier gas. temperature program (initial temperature of 80 °C was held for 1 min, followed by a linear ramp of 10 °C/min to 220 °C, 20 °C/min to of 280 °C and 5 °C/min up to 290 °C, which was held for 10 min. Peak integration was performed manually, and sterols were quantified from selected- ion monitoring analyses against internal standards using standard curves.

GC-MS analysis of sterol profile showed intracellular accumulation of 5,6p-EC associated with triol formation after treatment of C6 cells with 5,6p-EC (Figure 5). The cholestane triol is formed under the effect of ROS and/or in acidic environment enzymatically and is known as cytotoxic oxysterol. The characterization of cholesterol derivatives accumulated under the effect of 5,6p-EC has made it possible to highlight a predominant role of ROS in the antiproliferative effect of this molecule.

Recently, it has been proposed that increasing the intracellular level of ROS in cancer cells could become a promising approach to overcome cancer (40-42) by inducing autophagy that could also activate apoptosis signaling pathways in cancer cells (43-44). The results show that 5,6p-EC is prooxidant, inducer of mitochondrial and lysosomal dysfunction and activator of autophagy. In addition, a- tocopherol, DMF and MMF protect from cell death towards 5,6p-EC suggesting that oxidative stress is one of the key events leading to cell death.

Add to that, a very significant increase in the [LC3II I LC3I] ratio was observed with 5,6p-EC giving it a potent autophagy-inducing potential. Furthermore, Western blot analysis of PARP and caspase-3 protein did not reveal any cleavage in C6 cells treated with 5,6p-EC, showing that the cell death induced by this molecule in C6 cells is non-apoptotic cell death. This confirms the absence of apoptosis shown by Hoechst 33342 staining. The induction of non-apoptotic cell death associated with oxidative stress confirm the uniqueness cell death induced by 5,6p-EC. This would eventually allow the identification of new signaling pathways activated in this cell death. Due to its potential involvement in the metabolism of cholesterol, which is deregulated in glioblastoma, 5,6p-EC could be used in the antitumor metabolic therapy of glioblastoma.

The overall effect of 5,6p-EC would be to divert the stimulating activity of cholesterol, which accumulates in glioblastoma cells, into cytotoxic activity: the cholesterol sequestered in the cell would be oxidized under the effect of ROS induced by 5,6p-EC into cytotoxic oxysterols which would contribute to amplify cell death. This mechanism of action has important consequences for the specificity of the therapy. As normal cells have regulated and therefore low levels of cholesterol, they will not be affected by the toxicity of 5,6p-EC that would preferentially affect cholesterol-rich cells by generating cytotoxic oxysterols formed by auto-oxidation.

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