Methods for Determining the Oncogenic Condition of Cell, Uses Thereof, and Methods for Treating Cancer

Abstract

The invention relates to methods for detecting the oncogenic condition of cells, including step where the amount of the OCDO compound in said cells is measured, and to the uses thereof. The invention further relates to OCDO inhibitors for use in methods for treating cancer.

Claims

1-8. (canceled)

9. A method for treating a patient suffering from cancer comprising the steps of: a) obtaining a tumor cell sample from a patient suffering from cancer; b) measuring, in said tumor cell sample, the amount of 6-oxo-cholestane-3, 5-diol (OCDO); c) comparing the amount of OCDO measured in step b) with a reference value; d) providing: a poor prognosis for said patient if the amount of OCDO compound is significantly enhanced compared with said reference value; or a good prognosis for said patient if the amount of OCDO compound is lower than said reference value; and e) treating the patient with an anticancer treatment selected based on the prognosis provided in d).

10. The method for treating a patient suffering from cancer according to claim 9, wherein said method further comprises the steps of: f) obtaining a second tumor cell sample from the patient suffering from cancer; g) measuring, in said second tumor cell sample, the amount of 6-oxo-cholestane-3, 5-diol (OCDO); h) comparing the amount of OCDO measured in step g) with that measured in step b); i) assessing that: the treatment provided in step e) is effective if the amount of OCDO measured in step g) is lower than that measured in step b); or the treatment provided in step e) is ineffective if the amount of OCDO measured in step g) is higher than or equal to that measured in step b); and j) modifying the treatment if it is identified as ineffective in step i).

11. The method for treating a patient suffering from cancer according to claim 9, wherein the tumor cell sample is a liquid extract of the cells of a tumor sample obtained from said patient.

12. A method for treating a patient suffering from cancer comprising the steps of: a) obtaining a tumor cell sample from a patient suffering from cancer; b) measuring, in said tumor cell sample, the amount of 6-oxo-cholestane-3, 5-diol (OCDO); c) comparing the amount of OCDO measured in step b) with a reference value; d) providing: a good prediction of the therapeutic activity of OCDO inhibitors for said patient if the amount of OCDO compound is significantly enhanced compared with said reference value; and e) treating the patient with an OCDO inhibitor when a good prediction of the therapeutic activity is provided in d).

13. The method for treating a patient suffering from cancer according to claim 12, wherein the OCDO inhibitor is selected from: inhibitors of an enzyme involved in cholesterol biosynthesis, in particular lovastatin, Ro 48 8071, U18666A, AY-9944, triparanol, terbinafine and SKF-525A; cytochrome P450 inhibitors, lipoxygenases and antioxidants that are active on cholesterol epoxidation, such as ketoconazole and vitamin E; inhibitors of cholesterol epoxide hydrolase (ChEH) activity, in particular PBPE, PCPE, tesmilifene, dendrogenin A (DDA), tamoxifen, 4 hydroxytamoxifen, raloxifene, nitromiphene, clomiphene, RU 39411, BD-1008, haloperidol, SR 31747A, ibogaine, AC-915, rimcazole, amiodarone, trifluoroperazine, U18666A, AY 9944, triparanol, terbinafine and SKF-525A; inhibitors selected from the group consisting of: estrogen receptor antagonists; anti-estrogen membrane binding site (AEBS) ligands; ligands of -1 and -2 receptors and certain aminoalkyl sterols; intracellular cholesterol transport inhibitors; and enzyme inhibitors selected from the group consisting of progesterone and Ahr receptor antagonists.

14. The method for treating a patient suffering from cancer according to claim 13, wherein said OCDO inhibitor is Ro 48-8071 or ibogaine.

15. A method for identifying and treating an patient suffering from cancer comprising the steps of: a) obtaining a cell sample from a patient; b) measuring, in said cell sample, the amount of 6-oxo-cholestane-3, 5-diol (OCDO); c) comparing the amount of OCDO measured in step b) with a reference value; d) assessing that the patient has cancer if the amount of OCDO compound is significantly enhanced compared with said reference value; and e) treating said patient identified as having cancer at step d) with an anticancer treatment.

16. The method for identifying and treating a patient suffering from cancer according to claim 15, wherein the anticancer treatment consists of administering an effective amount of an OCDO inhibitor to said individual.

17. The method for identifying and treating a patient suffering from cancer according to claim 16, wherein the OCDO inhibitor is selected from: inhibitors of an enzyme involved in cholesterol biosynthesis, in particular lovastatin, Ro 48 8071, U18666A, AY-9944, triparanol, terbinafine and SKF-525A; cytochrome P450 inhibitors, lipoxygenases and antioxidants that are active on cholesterol epoxidation, such as ketoconazole and vitamin E; inhibitors of cholesterol epoxide hydrolase (ChEH) activity, in particular PBPE, PCPE, tesmilifene, dendrogenin A (DDA), tamoxifen, 4 hydroxytamoxifen, raloxifene, nitromiphene, clomiphene, RU 39411, BD-1008, haloperidol, SR 31747A, ibogaine, AC-915, rimcazole, amiodarone, trifluoroperazine, U18666A, AY 9944, triparanol, terbinafine and SKF-525A; inhibitors selected from the group consisting of: estrogen receptor antagonists; anti-estrogen membrane binding site (AEBS) ligands; ligands of -1 and -2 receptors and certain aminoalkyl sterols; intracellular cholesterol transport inhibitors; and enzyme inhibitors selected from the group consisting of progesterone and Ahr receptor antagonists.

18. The method for identifying and treating a patient suffering from cancer according to claim 17, wherein said OCDO inhibitor is Ro 48-8071 or ibogaine.

19. The method for identifying and treating a patient suffering from cancer according to claim 15, wherein the cell sample is a liquid extract of the cells of a sample obtained from said patient.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0097] FIG. 1: Measurement of the ChEH activity in MCF7 cells and in mouse hepatocytes. The cells are incubated in the presence of [.sup.14C]CE and then the lipids are extracted and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CE: 5,6-epoxycholestanol, CT: cholestane-3,5,6-triol, M: metabolite M.

[0098] FIG. 2: Kinetics of the ChEH activity in MCF7 cells. The MCF7 cells are incubated in the presence of [.sup.14C]CE (A) or of [.sup.14C]CE (B) for increasing times of 4 to 72 hours. The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CE: 5,6-epoxycholestanol, CT: cholestane-3,5,6-triol, M: metabolite M.

[0099] FIG. 3: Dose-dependent inhibition of the ChEH activity (3 days) in MCF7 cells by tamoxifen and by PBPE. The MCF7 cells are incubated in the presence of [.sup.14C]CE (A) or of [.sup.14C]CE (B) for three days in the presence of increasing concentrations of tamoxifen (A) or of PBPE (B). The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CE: 5,6-epoxycholestanol, CT: cholestane-3,5,6-triol, M: metabolite M.

[0100] FIG. 4: Conversion of CT into metabolite M. The MCF7 cells are incubated in the presence of [.sup.14C]CT (A) for 24, 48 and 72 hours. The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CE: 5,6-epoxycholestanol, CT: cholestane-3,5,6-triol. M: metabolite M.

[0101] FIG. 5: Mass spectrum of the metabolite C. The MCF7 cells are incubated in the presence of CE and then the lipids are extracted and then purified by HPLC and the fraction corresponding to the OCDO is analyzed by mass spectrometry.

[0102] FIG. 6: Analysis of OCDO production in healthy tissues. The cells originating from various mouse organs are incubated in the presence of [.sup.14C]CT (A) for 48 hours in the presence or absence of tamoxifen. The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CT: cholestane-3,5,6-triol. M: metabolite M.

[0103] FIG. 7: Effect of OCDO on cell proliferation in vitro. The cells of a human medullary thyroid carcinoma (TT cells) are cultured in the presence of increasing concentrations of OCDO ranging from 0.1 to 5 M, for 4 days. After this incubation time, the amount of cells is measured and the proliferative factor is determined.

[0104] FIG. 8: Effect of OCDO on tumor progression in vivo. TT cells are implanted on nude mice. The mice are treated either with the vehicle solvent (Solvent) or with 50 g/kg of OCDO by injection once a day, 5 days out of 7, for several weeks. The tumor volume is measured and reported on the graph as a function of time.

[0105] FIG. 9: Analysis of lymph nodes. The presence of tumor cells is investigated in the lymph nodes and compared between animals treated with the vehicle solvent (Solvent) or OCDO.

[0106] FIG. 10: Histomorphological analysis of the tumors. The tumors of the animals treated with the vehicle solvent (Solvent) or the OCDO are fixed and incubated in the presence of antibodies directed against a cytokeratin, calcitonin and an epithelial membrane antigen (EMA). The visualization is carried out by means of a streptavin-biotin-peroxidase complex followed by incubation with a diaminobenzidine solution, and then staining with hematoxylin is carried out. The presence of brown staining reveals the expression of the proteins labeled with the antibodies.

[0107] FIG. 11: Effect of OCDO on cytokine expression. THP1 cells are treated in vitro with 10 M of OCDO for 4 hours. The total RNAs of the cells are extracted and the expression of the genes encoding IL12 and IL10 is determined and reported on the figure.

[0108] FIG. 12: Analysis of OCDO by high performance liquid chromatography (HPLC). The OCDO is separated from the other oxysterols, such as 5,6-epoxycholestanol (CE), cholestane-3,5a,6-triol (CT) and 7-ketocholesterol (7-keto-Ch). UV detection at 210 nm is used.

[0109] FIG. 13: Calibration curve for OCDO by HPLC. Increasing amounts of from 0.1 to 5 g of OCDO are injected and analyzed by HPLC. The area of the peaks corresponding to OCDO is reported as a function of the mass of product injected. A correlation straight line is established from the graph.

[0110] FIG. 14: Analysis of the OCDO by gas chromatography (GC). A mixture of 5-cholestane and OCDO is trimethylsilylated and then analyzed by gas chromatography coupled to mass spectrometry (GC/MS). 3 peaks are obtained. The first (10.62 minutes) corresponds to 5-cholestane, the second (32.43 minutes) corresponds to OCDO having lost one molecule of H.sub.2O (OCDO-H.sub.2O), the third (36.68 minutes) corresponds to OCDO.

[0111] FIG. 15: Mass analyses of the peak emerging at 32.43 minutes by GC. The mass fragmentation profile reveals an OCDO dehydration product (M+472.3). The structure of the product obtained is represented.

[0112] FIG. 16: Mass analyses of the peak emerging at 36.68 minutes by GC. The mass fragmentation profile reveals an OCDO dehydration product (M+(CH.sub.3)546). The structure of the product obtained is represented.

[0113] FIG. 17: Calibration curve for OCDO by GC/MS. Increasing amounts of from 6.25 ng to 100 ng of OCDO are injected and analyzed by GC/MS. The area of the peaks corresponding to OCDO is reported as a function of the mass of product injected. A correlation straight line is established from the graph.

[0114] FIG. 18: Inhibition of tumor growth by tamoxifen in vivo. Mammary cells of TS/A type are implanted in BALB/c mice. The mice are treated with tamoxifen (Tamoxifen) or with the vehicle solvent (vehicle). The volume of the tumors is measured over time.

[0115] FIG. 19: GC analysis of the OCDO content of the tumors. The tumors of the animals treated or not treated with tamoxifen are extracted. The extracts are analyzed by GC/MS. The GC profile of the extracts of the animals treated with the vehicle solvent (A) reveals the presence of peaks corresponding to the OCDO and to its dehydration product (OCDO-H.sub.2O).

[0116] FIG. 20: MS analysis of the GC peaks corresponding to OCDO and OCDO-H.sub.2O. The mass analysis confirms the structure of the compounds present in the peaks obtained by GC.

[0117] FIG. 21: Inhibition of OCDO production in MCF7 cells by ChEH inhibitors. The MCF7 cells are incubated in the presence of [.sup.14C]CE for three days in the presence of the vehicle solvent (T), of cholesterol epoxide (CE), of tamoxifen (Tam), of PBPE (PBPE), of raloxifene (Ral), and of tesmilifene (DPPE). The lane marked CE corresponds to a deposit of [.sup.14C]CE used as migration standard. The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CE: 5,6-epoxycholestanol, CT: cholestane-3,5,6-triol.

[0118] FIG. 22: Inhibition of OCDO production in MCF7 cells by ketoconazole, a general cytochrome P450 inhibitor. The MCF7 cells are incubated in the presence of [.sup.14C]CE for three days in the presence of the vehicle solvent (control) or of 10 M of ketoconazole. The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography and the amount of CE and of OCDO is quantified.

[0119] FIG. 23: Inhibition of OCDO production in MCF7 cells by an aminoalkyl sterol (DDA). Lane 1 corresponds to a deposit of [.sup.14C]CE used as migration standard. Lane 2 corresponds to a deposit of [.sup.14C]CT used as migration standard. The MCF7 cells are incubated in the presence of [.sup.14C]CE for 48 hours in the presence of the vehicle solvent (lane 3), of 0.1 M of DDA (lane 4) and of 1 M of DDA. The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CE: 5,6-epoxycholestanol, CT: cholestane-3,5,6-triol.

[0120] FIG. 24: Inhibition of OCDO production in MCF7 cells by intracellular cholesterol transport modulators and AhR receptor modulators: the MCF7 cells are incubated in the presence of [.sup.14C]CT for 24 hours in the presence of the vehicle solvent (lane 1), of progesterone (lane 2), of U18666A (lane 3), of TCDD (lane 4), of benzo(A)pyrene (lane 5), of resveratrol (lane 6) and of PDM2 (lane 7). The lipids are extracted from the cells and the sterols are analyzed and separated by silica plate thin-layer chromatography. The chromatography plate is visualized by autoradiography. CT: cholestane-3,5,6-triol.

EXAMPLE 1: OCDO IS A MARKER FOR THE TUMOR STATE OF A CELL

[0121] Using the method described in example 4 hereinafter, the presence of OCDO was tested on numerous tumor cell lines in order to establish that this marker can be observed in various cell types in humans, rats or mice. OCDO production was detected in each of the tumor cell lines tested, as indicated in the following table:

TABLE-US-00001 OCDO Cell line Type production MCF7 Human mammary carcinoma + MCF7/TamR Tamoxifen-resistant human mammary + carcinoma MDA-MB-231 Human mammary carcinoma + TS/A Murine mammary carcinoma + A-549 Human lung carcinoma + B16-F10 Murine melanoma + SK-MEL-28 Human melanoma + U-937 Human myeloid leukemia + J 774 Murine myeloid leukemia + THP.1 Human acute monocytic leukemia + HT-29 Human colon carcinoma + HeLa Human uterine carcinoma + C6 Rat glyoma + SK-N-SH Human neuroblastoma + Saos-2 Human osteosarcoma + P19 Murine embryonic carcinoma + TT Human medullary thyroid carcinoma +

[0122] Having noted that OCDO is found in all the tumor cells tested, the applicants concluded that OCDO constituted a marker for the tumor state of the cells.

[0123] However, as a preliminary study, and in a manner analogous to what was done in assays 1 to 5 above, the conversion of the CE compound was evaluated in cells of various healthy mouse tissues under conditions which made it possible to detect OCDO (see the result in FIG. 6). Said cells were incubated with the [.sup.14C]CE compound and the radioactive spots which appear with (+) or without () incubation with tamoxifen were observed by silica thin-layer chromatography. It is noted that, if tamoxifen is present, the CT compound no longer appears, which corresponds well to the inhibition of ChEH; in the absence of incubation with tamoxifen, the CT compound appears, but no presence, in the vicinity of the CE compound, of a spot corresponding to the presence of OCDO is noted.

[0124] It is therefore clear that OCDO is not produced in a normal tissue; its detection therefore clearly constitutes an element for predicting the tumor state of the cells; OCDO is a marker for the oncogenic state of a cell.

EXAMPLE 2: OCDO IS CARCINOGENIC

[0125] On the basis of the assays described above and of example 1, the applicants thought, according to the invention, that the presence of OCDO in the cells in a tumor situation could be linked to the fact that this molecule could by itself have a carcinogenic potential.

[0126] Such a potential has never been reported previously in the literature.

[0127] Cholesterol does not have mutagenic properties. The OCDO precursors and the cholesterol epoxide epimers have been mentioned as weak mutagens in mammalian cells (Peterson A R, Peterson H, Spears C P, Trosko J E and Sevanian, Mutation Research, vol. 203, p. 355-366, 1988). Sterol epoxides have not shown any mutagenic properties in the Ames test carried out on bacteria (Smith L L et al., Mutation Research, vol. 68, p. 23-30, 1979; Ansari GAS et al., vol. 20, p. 35-41, 1982). These observations were recently confirmed (Cheng Y W et al., Food and Chemical Toxicology, vol. 43, p. 617-622, 2005). Admittedly, structural homologies of OCDO with tumor promoters such as TPA (12-tetradecanoylphorbol 13-acetate) (Endo Y. et al., Chem. Pharm. Bull. (Tokyo), vol. 42 (3), p. 462-469, 1994) have been found. However, no-one has described or even suggested that OCDO could have PKC-activating properties or tumor-promoting properties in laboratory animals; it has only been shown that this molecule binds to a phorbol ester-binding protein (Endo Y., Biochem. Biophys. Res. Comm., vol. 194, p. 1529-1535, 1993).

[0128] From the cellular point of view, it has only been indicated that OCDO appears: [0129] 1) to inhibit rosette formation by T lymphocytes originating from human serum (Streuli R. A. et al., J. Immunol., vol. 123 (6)n, p. 2897-2902); [0130] 2) to inhibit leukocyte mobility (Gordon L., Proc. Natl. Acad. Sci. USA, vol. 77(7), p. 4313-4316, 1980); [0131] 3) to induce NK cell toxicity in mice (Kucuk O. et al., Cell. Immunol., 139, p. 541-549, 1992); [0132] 4) to inhibit the cytolytic activity produced by T lymphocytes in mice (Kucuk O et al., Lipids, vol. 29(9), p. 657-660, 1994), these effects being observed at concentrations between 1 and 20 M.

[0133] Through the present example 2, the applicants established, in the context of the present invention, that OCDO has effects on tumor development, which were not in any way suggested to those skilled in the art by the prior art.

a) In this example 2, the human medullary thyroid carcinoma cell line TT (American Tissues and Cells Collection) was used. This line is cultured in an F12K medium modified by Kaighn (sold by the company Invitrogen), containing 10% by weight of fetal calf serum and 2.5 ml of a solution of antibiotics (penicillin/streptomycin) at 50 IU/g. The OCDO tested comes from the company Steraloids. In order to study in vitro the effect of OCDO on cell growth, the TT cells are seeded on to six-well plates (200 000 cells per well) in an F12K medium as defined above. The TT cells are treated every two days either with a solvent (ethanol at 0.1% in PBS phosphate buffer) or with amounts of OCDO in the solvent resulting in concentrations of 0.1, 1, 2.5 or 5 M. The cells are counted 4 days after seeding. As is seen in FIG. 7, the proliferation of the TT cells is increased in a concentration-dependent manner by the treatment with OCDO, when compared, as reference, with the proliferation of the cells treated with the solvent. At the OCDO concentration of 5 M, a proliferation induction factor of 1.5 is measured after 4 days of treatment; the induction of proliferation is therefore established in vitro for OCDO concentrations of between 100 nM and 5 M. The proliferation-stimulating factor is comparable to that of estrogens, which are molecules that have mitogenic properties on mammary tumor cells (see: Medina et al., J Pharmacol Exp Ther, vol. 319, 2006: p. 139-149).
b) The applicants also studied in vivo the effect of OCDO on tumor development. For injection into animals, the TT cells defined above in this example under a) are recovered with trypsin, washed twice and suspended in a PBS phosphate buffer. The TT cells (approximately 410.sup.6 cells/0.1 ml) are then injected subcutaneously into the flank of 6-week-old Swiss nude nu/nu female mice (supplied by Charles River). The animals are treated subcutaneously once a day, on 5 days out of 7, either with OCDO at the dose of 50 g/kg (treated group) or with the solvent (control group) over a period of 110 days (the solvent used is 0.1% ethanol in phosphate buffer (PBS)). The animals (5 or 10 mice per group) are monitored regularly in order to measure tumor development. The volume of the tumors is calculated according to the formula Ll.sup.20.5, where L is the length and l is the width of the tumor. FIG. 8 represents the volume V of the tumors as a function of treatment time t. The treatment with OCDO significantly accelerates the tumor growth; the tumor volume in the animals treated with OCDO is almost three times larger than for the control animals treated with the solvent.

[0134] The animals were euthanized after 75 days of treatment and the tumor and the various organs were removed in order to be analyzed histologically. Twice as much invasion of the lymph nodes (LN) was observed in the animals treated with OCDO compared with the animals treated with the solvent (see FIG. 9).

[0135] A histomorphological analysis was performed. To do this, the tumors of the mice treated with OCDO or with the solvent and also various organs (lymph nodes, lung and liver) are removed, fixed in 10% buffered neutral formalin and embedded in paraffin blocks. For these analyses, the sections are stained with hematoxylin and eosin. The immunolabeling is carried out with antibodies directed against various human antigens associated with medullary thyroid carcinomas. The antibodies used are the anti-calcitonin polyclonal antibody (Dako SAS, Trappes, France, 1:1000), the anti-cytokeratin monoclonal antibody (Dako clone AE1/AE3, 1:50) and the anti-epithelial membrane antigen (EMA) monoclonal antibody (Dako clone E29, 1:50). The immunolabeling of the paraffin sections is preceded by an antigen recovery technique by heating in a citrate buffer (10 mM, pH 6) either twice for 10 minutes in a microwave oven (750 W) for the anti-CEA antibody, or in a waterbath at 95 C. for 40 minutes for the anti-calcitonin and anti-EMA antibodies.

[0136] After incubation with the antibodies defined above, the sections are immunolabeled with the streptavidin-biotin-peroxidase complex (StreptABComplex/HRP, Dako) followed by a chromogenic solution of diaminobenzidine (DAB), and are then stained with hematoxylin.

[0137] The negative controls are performed by incubation in a buffer solution not containing the primary antibody. The results obtained by means of this histological analysis are represented in FIG. 10 for each of the three antibodies used. It is noted that the tumors derived from the animals treated with OCDO are indeed human medullary thyroid carcinomas, which confirms the invasion of the lymph nodes by cells derived from the tumor.

[0138] The applicants therefore established that OCDO has a mitogenic activity in vivo by stimulating implanted tumor growth in laboratory animals.

EXAMPLE 3: OCDO STIMULATES IL10 AND REDUCES IL12, WHICH EXPLAINS ITS CARCINOGENIC ACTION

[0139] The applicants, having established, according to the invention, that OCDO stimulates the invasive capacity of cancer cells in vivo, confirmed this effect of OCDO by studying, in vitro, the expression of cytokines on THP1 cells treated in vitro with OCDO.

[0140] The THP1 cells (human myeloid cell line supplied by ATCC) are cultured with a culture medium of DMEM type (Dulbecco's modified eagle medium) supplemented with 10% of fetal calf serum and a mixture of antibiotics. The THP1 cells are treated with 10 M of OCDO for 4 hours. The RNAs are extracted according to a conventional procedure. The complementary DNAs are produced, and then used to measure the expression of the genes of interest. The expression of the genes encoding interleukin 12 (IL12) and interleukin 10 (IL10) were studied, these interleukins representing a pair of cytokines with antagonistic properties. IL12 is an immunostimulatory cytokine, whereas IL10 is an immunosuppressive cytokine. The expression ratio of these two cytokines makes it possible to evaluate the immunosuppressor or immunostimulatory potential of the cell and its ability to promote tumor progression or, on the contrary, to slow it down.

[0141] The primers used correspond to the human sequences of IL10 and of IL12 and are the following:

TABLE-US-00002 IL10sense: (SEQIDNO:1) AAA-CCA-AAC-CAC-AAG-ACA-GAC, IL10anti-sense: (SEQIDNO:2) GCT-GAA-GGC-ATC-TCG-GAG, IL12sense: (SEQIDNO:3) CTA-TGG-TGA-GCC-GTG-ATT-GTG, IL12anti-sense: (SEQIDNO:4) TCT-GTG-TCA-TCC-TCC-TGT-GTC.

[0142] The results are represented in FIG. 11: OCDO stimulates the expression of the immunosuppressive cytokine IL10 and reduces the expression of the cytokine IL12. This mechanism makes it possible to explain the stimulation by OCDO of the invasive capacity of tumor cells noted via example 2.

EXAMPLE 4: ASSAYING OF OCDO

A) Assaying by HPLC

[0143] The OCDO was separated and assayed by a high performance liquid chromatography HPLC method (95 MeOH/5 H.sub.2O; 0.7 ml/min; column Ultrasep ES 6 m of 2504, C18 (Bishoff, Leonberg, Germany)). The chromatograph is an apparatus from the company Perkin Elmer; it comprises a series 200 pump and a diode array detector of type 200.

[0144] The apparatus is equipped with a PC computer, which uses the Turbochrome) for apparatus control and data processing.

[0145] A cell extract of cultured MCF7 tumor cells is prepared as indicated in assay 1: approximately 60 million cells are lyzed so as to obtain 25 l of liquid extract after centrifugation for 10 min at 1200 rpm. 20 l of this extract are passed through the column of the chromatograph.

[0146] The resulting chromatogram is provided in FIG. 12: it is seen that the OCDO is separated from the CT, from the CE and from the keto-cholesterol (7-keto-Ch). The retention time of the OCDO is 19 min.

[0147] A calibration is performed in order to link the measurement of the surface area of the OCDO peak thus obtained and the weight of the OCDO contained in the sample.

[0148] This calibration is carried out using ethanolic solutions of OCDO sold by the company Steraloids, of increasing concentrations. A fixed volume of 20 l of sample is used. The following amounts of OCDO were injected: 80 ng, 0.4 g, 0.8 g, 4 g and 8 g. The area of the peaks corresponding to the OCDO was measured by integration using the Turbochrome software; these values (y) were reported on the graph of FIG. 13 as a function of the corresponding OCDO masses (x). A suitable correlation straight line is obtained (y=69430x+1381; r.sup.2=0.999; n=63; p<0.0001; x in g of OCDO; y in V.Math.s) making it possible to quantify the OCDO for masses of between 40 ng and 80 g. This method makes it possible to quantify the OCDO for amounts of between 0.1 and 5 g of molecules.

B) Assaying by Gas Chromatography Coupled with Mass Spectrometry (GC/MS)

[0149] A mixture of 5-cholestane and OCDO is trimethylsilylated according to the method described by Kedjouar (see Kedjouar et al., J Biol Chem, 2004, vol. 279, No. 32, p. 34048-61). The sample is treated with 20 a mixture of N,O-bis(trimethylsilyl)-trifluoroacetamide/pyridine (50:50, v/v) for 30 min at a temperature of 60 C. The reagents are evaporated under a stream of nitrogen and the trimethylsilylated (TMS) derivatives are dissolved in hexane. These GC/MS analyses are carried out on a Hewlett Packard system apparatus type 4890 equipped with an RTX-50 silica capillary column (30 m0.32 mm internal diameter, film of thickness 0.1 m; Restek). The oven temperature was programmed at 230 C. for 1 minute, then from 230 to 240 C. at a rate of increase of 1 C. per minute for 10 minutes, from 240 C. to 250 C. at a rate of increase of 3 C. per minute, then up to 290 C. at a rate of increase of 45 C./min then to 330 C. in 1 minute.

[0150] The injector and the detector were at 310 C. and 340 C., respectively. The GC profile obtained is represented in FIG. 14. Three peaks were obtained. The first corresponds to 5-cholestane, which has a retention time of 10.62 minutes. The second corresponds to a retention time of 32.43 minutes and gives, in analysis by mass spectrometry with electron impact fragmentation, a result which corresponds to an OCDO dehydration product (M+(472.3)), M+ minus a CH.sub.3 group and M+ minus 2 CH.sub.3 groups (see FIG. 15). Finally, the last peak (retention time of 36.68 minutes) corresponds to the OCDO and its mass fragmentation profile is given in FIG. 16. This profile determines the masses of 546.2 (M+ minus a CH.sub.3), 531.2 (M+ minus two CH.sub.3) and 472.2 (M+ minus H.sub.2O and minus an OTMS group).

[0151] The calibration of the method is carried out using ethanolic solutions of OCDO of increasing concentrations. The following amounts of OCDO were injected: 6.25 ng, 12.5 ng, 50 ng and 100 ng. Integration of the area of the peaks corresponding to the OCDO for these various amounts of OCDO made it possible to establish a calibration curve for quantifying the OCDO. This method makes it possible to assay amounts of OCDO of between 5 and 200 ng (see FIG. 17).

EXAMPLE 5: INHIBITION OF OCDO PRODUCTION BY TAMOXIFEN IN VIVO

[0152] As has already been shown in assay 2 (FIG. 3A) that the addition of tamoxifen to MCF7 tumor cell cultures blocks OCDO production in these cells. It will be shown hereinafter that tamoxifen administered in vivo also causes an inhibition of OCDO production.

[0153] The assaying techniques used in example 4 were used for measuring the modulation of OCDO on tumors implanted in mice.

[0154] A cell culture of TS/A cells was first performed. These TS/A cells are mouse mammary adenocarcinomas (see Nanni P et al., Clin. Exp. Metastasis, 1983 October-December; 1(4): 373-80). The cells are cultured, at 37 C. under 5% CO.sub.2, in a DMEM medium supplemented with 2 g/liter of sodium carbonate, 1.2 mM of glutamine (pH 7.4 at 23 C.), 5% of fetal bovine serum (Gibco) and 2.5 ml of antibiotics per liter of medium (penicillin/streptomycin).

[0155] The TS/A cells are recovered with trypsin, washed twice and suspended in PBS buffer. The TS/A cells (approximately 410.sup.6 15 cells/0.1 ml) are then injected subcutaneously into the flank of 6-week-old female BALB/c mice (supplied by Charles River). The animals are treated for 27 days after the implantation of the tumors, intratumorally once a day over a period of 37 days, either with tamoxifen at a concentration of 10 M for injection volumes of 100 l (group treated with tamoxifen), or with the vehicle solvent (ethanol at 0.1% in PBS phosphate buffer) (control group). The animals (10 mice per group) are monitored regularly for tumor development. The volume of the tumors is calculated according to the following formula: Ll.sup.20.5 (L is the length and l is the width of the tumor). The experiment was reproduced twice. The results are given in FIG. 18 (the arrow indicates the beginning of the treatment).

[0156] An inhibition of tumor growth in the mice treated with tamoxifen was thus qualitatively observed in FIG. 18.

[0157] In order to obtain quantitative information on the inhibition of OCDO, the experiment was reproduced under the same conditions and the tumors were removed on day 28. 5 volumes (relative to the mass removed) of buffer (50 mM Tris-HCl, 150 mM KCl, pH=7.4) are added. The tumors are ground and the homogenates are centrifuged for 5 minutes at 2500 rpm at 4 C. The supernatants are recovered and then 1 volume of methanol (relative to the volume of supernatant) and 2 volumes of chloroform are added. After centrifugation (to separate the phases), the organic phase is recovered. The organic phase is evaporated to dryness and then the residue is taken up with 0.5 ml of chloroform. The organic extracts are filtered on 0.5 ml silica cartridges. The polar sterols are eluted sequentially:

1) 0.5 ml of a hexane/chloroform mixture (1/1),
2) 0.5 ml of chloroform,
3) 0.5 ml of ethyl acetate and methanol.

[0158] The addition of a .sup.14C-labeled OCDO external standard during the extraction and purification phases makes it possible to establish a recovery yield of 866%. The ethyl acetate fractions are evaporated, silylated (50 l 1/1 acetonitrile/BSTFA) and then analyzed by GC/MS (2 l) according to the conditions described above.

[0159] FIG. 19 shows two chromatograms prepared in the gas phase as indicated in example 4, corresponding:

A) to an extract of tumor originating from a control animal: the tumor had a mass of 2 g;
B) to an extract of tumor originating from an animal treated with tamoxifen: the tumor had a mass of 1.45 g.

[0160] A significant decrease in the peaks originating from the OCDO in the tumor of the treated mice (B) compared with that of the nontreated mice (A) is observed in FIG. 19. The mass spectrometry analysis confirms the structure of the molecules in the peaks of interest (see FIG. 20).

[0161] The results thus obtained, in terms of numbers, have been given below, the control group having been previously defined, and the values associated with the control have been valued at 100 with coefficients which were also applied to the results relating to tamoxifen.

TABLE-US-00003 Day 28 Day 37 Tumor size OCDO Tumor size OCDO (% (% (% (% Molecule control) control) control) control) Nontreated 100 100 100 100 control Tamoxifen 73.5 8 19.2 4 46.2 7 8.4 4

[0162] It is seen that, in vivo, tamoxifen significantly inhibits OCDO.

[0163] The fact that tamoxifen exerts an antitumor activity while at the same time inhibiting, in vivo, the production of a tumor-promoting oxysterol shows that OCDO plays a role in the effects of tamoxifen.

EXAMPLE 6: INHIBITION OF OCDO PRODUCTION BY PBPE IN VIVO

[0164] PBPE is a compound of which the antiproliferative effect was established by in vitro experiments (see the reference cited in assay 2 and also the publication Payre B. et al., Mol. Cancer Ther., 7(12), 3707-3717). In vivo results analogous to those present in example 5 for tamoxifen were determined for PBPE. The protocol used is strictly identical to that which was detailed in example 5 for the treatment with tamoxifen, with the only difference that the daily intratumor injections are carried out at a concentration of 40 M for PBPE for injection volumes of 100 l. The results are collated in table 5 below:

TABLE-US-00004 Day 28 Day 37 Tumor size OCDO Tumor size OCDO (% (% (% (% Molecule control) control) control) control) Nontreated 100 100 100 100 control PBPE 71.6 7 22.5 5 54.5 8 9.6 5

[0165] It was thus determined in vivo that PBPE both inhibits OCDO and causes tumors to regress.

EXAMPLE 7: INHIBITION OF OCDO PRODUCTION BY PBPE ON VARIOUS TUMOR CELLS

[0166] Assay 2 provided above showed that cell extracts of MCF7 tumor cells incubated for 3 days in PBPE solutions lead to the observation of an inhibition of the metabolite that was identified as being OCDO.

[0167] An analogous experiment was reproduced with different cell lines, with the cells tested being incubated for 48 hours in a 40 M solution of PBPE. It was noted that the OCDO was 100% inhibited for all the lines in the following table that were tested:

TABLE-US-00005 Inhibition of Cell line Type OCDO production MCF-7 Human mammary carcinoma 100% MDA-MB-231 Human mammary carcinoma 100% A-549 Human lung carcinoma 100% B-16-F10 Murine melanoma 100% U-937 Human leukemia 100% HT-29 Human colon carcinoma 100% HeLa Human uterine carcinoma 100% C6 Rat glyoma 100% SH-N-SH Human neuroblastoma 100% Saos-2 Human osteosarcoma 100% P19 Murine carcinoembryonic 100% line TT Human medullary thyroid 100% carcinoma

[0168] Generally, in order to establish the percentages of OCDO inhibition from the thin-layer chromatography (TLC) plates obtained as in assay 2, the radioactive metabolites are identified and quantified on the basis of said plates using a europium-sensitive plate of GP Phosphor screen type (GE Healthcare) and a Storm 840 phosphorimagor (GE Healthcare). The proportion of radiolabeled oxysterols is determined on the autoradiogram obtained by densitometry using the Image Quant 5.2 software. The percentage is calculated on the basis of the ratio between the amount of oxysterols quantified divided by the sum of the amounts of oxysterols (CEE+CE+CT+OCDO). Since the CEE (CE ester) originates exclusively from the CE, the percentage CE is calculated on the basis of the ratio (CE+CEE)/(CE+CEE+CT+OCDO).

EXAMPLE 8: OCDO IS INHIBITED WHEN CHEH IS INACTIVE

[0169] As was previously established in this patent application, it is known that tumor cells produce OCDO, which implies that the ChEH hydrolase is active since it enables the conversion of CE to CT, which is the obligatory change for the production of OCDO. As will be established in table 1 given later in this example, tamoxifen, PBPE, raloxifene and DPPE are ChEH inhibitors. Assay 2 given above was therefore completed by measuring the amounts of the products present when the MCF7 tumor cells are incubated as indicated in detail in assay 2. The calculation of the percentages of CE, CT and OCDO was carried out as indicated in example 7. The results are given in FIG. 21, which shows a thin-layer chromatography plate on which the lanes correspond to incubations of the cells with the vehicle solvent T (0.1% ethanol in PBS buffer), with CE- and with four ChEH inhibitors, the identifiers of which are given at the bottom of the lanes. The numerical values obtained are collated in the table below:

TABLE-US-00006 Compounds % CE % OCDO % CT EtOH (T) 0 73.1 26.9 CE 10 M 31.5 27 41.5 Tam 2.5 M 90.3 6.9 2.8 PBPE 10 M 89.2 6.2 4.6 Ral 10 M 96.5 0.5 3 DPPE 1.5 M 80.7 8.8 10.5 T = control DPPE = N,N-diethyl-2-(4-benzylphenoxy)ethanamine Ral = raloxifene

[0170] It is noted that inhibition of ChEH indeed leads to inhibition of OCDO, the CT product being very reduced in amount.

[0171] In fact, the formation of OCDO in a cell depends on several parameters: [0172] 1. the presence of cholesterol, [0173] 2. the presence of CE, [0174] 3. the presence of cholesterol epoxide hydrolase (ChEH), [0175] 4. the presence of CT, [0176] 5. the transport of CT to the zone where CT is oxidized to OCDO, [0177] 6. the presence and the activity of the enzyme responsible for the oxidation of CT to OCDO.

[0178] In order to establish that the link that exists between, on the one hand, the ability of various compounds to inhibit OCDO in an MCF7 tumor cell and, on the other hand, their quality as a ChEH inhibitor, is well-founded, the inhibition coefficients Ki for ChEH relating to a certain number of products was calculated and the production of OCDO by MCF7 cells after incubation of the cells with the same products was measured according to the protocol defined in assay 2 of the present patent application. All of the results are given in tables 1 and 2. Based on these two tables, it appears that any inhibitor of an enzyme involved in cholesterol biosynthesis causes a decrease in the cholesterol that can be used for OCDO production. Inhibitors of enzymes from hydromethylglutaryl coenzyme A synthetase (HMG CoA synthetase) to 7-dehydrocholesterol reductase and 24-dehydrocholesterol reductase will in particular be noted.

[0179] An experiment was carried out among this category of inhibitors, namely with an HMG-CoA reductase inhibitor, such as lovastatin, used at a concentration of 10 M: it was noted that, after incubation of MCF7 cells according to the protocol given in assay 2 and by bringing the control to the value 100, an OCDO production by the MCF7 cells of less than 1 is obtained.

TABLE-US-00007 TABLE 1 Molecule K.sub.i ChEH (nM) AEBS ligands PBPE 26.8 6 PCPE 34.7 8 Tesmilifene 62.4 3 DDA 1250.4 6 Estrogen receptor Tamoxifen 33.6 8 modulators 4OH-Tamoxifen 145.3 4 Raloxifene 35.6 4 Nitromiphene 17.7 6 Clomiphene 9.0 2 RU 39,411 155.2 8 Sigma receptor BD-1008 98.7 9 ligands Haloperidol 18067 14 SR-31747A 6.2 2 Ibogaine 2150 11 AC-915 3527 9 Rimcazole 2325 8 Amiodarone 733.1 9 Trifluoroperazine 135.4 7 Cholesterol Ro 48-8071 88.9 5 biosynthesis U-18666A 90.3 5 inhibitors AY-9944 649 6 Triparanol 39.5 3 Terbinafine 9105 33 SKF-525A 1904 11

TABLE-US-00008 TABLE 2 OCDO production by Molecule MCF-7 (concentration in M) (% control) Control 100 AEBS ligands PBPE (1) <1 PCPE (1) <1 Tesmilifene (1) <1 DDA <1 Estrogen receptor Tamoxifen (1) <1 modulators 4OH-Tamoxifen (1) <1 Raloxifene (1) <1 Nitromiphene (1) <1 Clomiphene (1) <1 RU 39,411 (5) <1 receptor BD-1008 (1) <1 ligands Haloperidol (100) 42 SR-31747A (1) <1 Ibogaine (5) 25 AC-915 (10) 16 Rimcazole (5) 12 Amiodarone (5) 8 Trifluoroperazine (1) <1 Cholesterol Ro 48-8071 (10) <1 biosynthesis U-18666A (1) <1 inhibitors AY-9944 (5) <1 Triparanol (1) <1 Terbinafine (10) <1 SKF-525A (10) <1

[0180] The products which appear in tables 1 and 2 and which have not been previously identified in the present text are defined by their chemical name in the following list: [0181] PCPE: 1-(2-(4-(2-phenylpropan-2-yl)phenoxy)ethyl)-pyrrolidine; [0182] tesmilifene: 2-(4-benzylphenoxy)-N,N-diethylethanamine; tamoxifen: 2-[4-[(Z)-1,2-di(phenyl)but-1-enyl]phenoxy]-N,N-dimethylethanamine; [0183] 4OH-tamoxifen: 4-[(Z)-1-[4-(2-dimethylaminoethyloxy)-phenyl]-2-phenylbut-1-enyl]phenol; [0184] raloxifene: [6-hydroxy-2-(4-hydroxyphenyl)-1-benzothiophen-3-yl]-[4-(2-piperidin-1-ylethoxy)phenyl]methanone; [0185] nitromiphene: 1-[2-[4-[(Z)-1-(4-methoxyphenyl)-2-nitro-2-phenylethenyl]phenoxy]ethyl]pyrrolidine; [0186] clomiphene: 2-[4-[(Z)-2-chloro-1,2-di(phenyl)ethenyl]-phenoxy]-N,N-diethylethanamine; [0187] RU 39411: 11-[4-N,N-[diethylaminoethoxy]phenyl]estra-1,3,5(10)triene-3,17-diol. [0188] BD-1008: N-(3,4-dichlorophenethyl)-N-methyl-2-(pyrrolidon-1-yl)ethanamine; [0189] haloperidol: 4-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-1-(4-fluorophenyl)butan-1-one; SR-31747A: (E)-N-(4-(3-chloro-4-cyclohexylphenyl)but-3-enyl)-N-ethylcyclohexanamine; [0190] AC915: N-(3,4-dichlorophenethyl)-N-methyl-2-(pyrrolidon-1-yl)ethanamine; [0191] rimcazole: 9-[3-[(3S,5R)-3,5-dimethylpiperazin-1-yl]propyl]carbazole; [0192] amiodarone: (2-butyl-1-benzofuran-3-yl)-[4-(2-diethylaminoethyloxy)-3,5-diiodophenyl]methanone; [0193] trifluoroperazine: 10-[3-(4-methylpiperazin-1-yl)propyl]-2-(trifluoromethyl)phenothiazine; [0194] RO 48-8071: (4-(6-(allyl(methyl)amino)hexyloxy)phenyl) (4-bromophenyl)-methanone; [0195] U-18666A: 3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; [0196] AY-9944: trans-1,4-bis(2-chlorobenzaminomethyl)-cyclohexane; [0197] triparanol: 2-(4-chlorophenyl)-1-(4-(2-diethylamino)-ethoxy)phenyl)-1-p-tolylethanol; [0198] terbinafine: (E)-N,3,6,6-tetramethyl-N-(naphthalen-1-ylmethyl)hept-2-en-4-yn-1-amine; [0199] SKF-525A: 2-diethylaminoethyl-2,2-diphenylpentanoate.

[0200] The inhibitors which were the subject of experiments having results in tables 1 and 2 include estrogen receptor modulators, anti-estrogen membrane binding site (AEBS) ligands, sigma-1 and -2 receptor ligands, and inhibitors of cholesterol biosynthesis from squalene synthetase up to 7- and 24-dehydrocholesterol reductase. All these inhibitors have one thing in common, namely that of being AEBS ligands.

[0201] The protocols used for measuring the inhibition coefficients Ki of table 1 are given in detail hereinafter:

[0202] a) Ki for the AEBSs

[0203] The Ki is the inhibition constant corresponding to the molecule of interest and is measured in the following way: rat liver microsomes are incubated with a concentrations of 2.5 nM of tritiated tamoxifen (supplied by the company GE Healthcare) and increasing concentration of inhibitor of between 0.01 and 1000 M under the conditions described in the following publication: Poirot M. et al., Bioorg Med Chem 2000, vol. 8(8), p. 2007-2016. The IC50 values correspond to the concentration of inhibitor required to inhibit 50% of the activity of the ChEH; they are determined using a GraphPad Prism (version 4) data processing program. The Ki values are calculated using the Cheng-Prussof equation (Cheng and Prussof, Biochem Pharmacol, 1973, vol. 22(23), pages 3099-3108). The Ki is expressed according to the equation: Ki=[IC50](1+(tritiated tamoxifen])/Kd). The concentration of tritiated tamoxifen is 2.5 nM and the dissociation constant at equilibrium of the tritiated tamoxifen for AEBS is 2 nM.

[0204] b) Determination of the Ki for ChEH:

[0205] 150 g of rat liver microsome proteins are incubated in the presence of 2 concentrations of [.sup.14C]CE with increasing concentrations of inhibitors of between 0.01 and 1000 M under the conditions described above for measuring the ChEH activity. The Ki is measured as the projection on the x-axis of the intersection of the lines obtained by reporting on a graph the values of 1/V as a function of 1/S for ChEH, as determined by the Dixon method (Dixon M, Biocheml Jl, 1953, vol. 55(1), p. 170-171).

EXAMPLE 9: INHIBITION OF OCDO BY A CYTOCHROME P450 INHIBITOR

[0206] Cholesterol epoxidation can be produced by self-oxidation of cholesterol with oxygen in the air, under the action of enzymes such as cytochromes P450 or lipoxygenases (see Schroepfer G. Jr, Physiological Reviews, vol. 80, No. 1, p. 361-554, 2000).

[0207] An inhibition of the production of the epoxysterol CE and of its derivatives, which are CT and OCDO, was noted when using a general cytochrome P450 inhibitor, namely ketoconazole (see FIG. 22).

[0208] The protocol for this experiment is the same as that described in example 7.

EXAMPLE 10: INHIBITION OF CHEH BY AN AMINOALKYL STEROL

[0209] For this experiment, an aminoalkyl sterol included in French patent 2 838 741 (and also see: de Medina et al., J Med Chem: 2009, vol. 52, No. 23, p. 7765-7777), having the formula: 6-N-[2-(3H-imidazol-4-yl)ethylamino]cholestane-3,5-diol (DDA), was chosen. MCF7 tumor cells were incubated with [.sup.14C]CE (10 mCurie/mmol, 0.6 M) for 48 hours in the presence or absence of the abovementioned aminoalkyl sterol (at the concentrations of 0.1 and 1 M).

[0210] A thin-layer chromatography was carried out, the plate of which has been reproduced in FIG. 23. On this plate, the incubation is carried out, for lane 1, with [.sup.14C]CE; for lane 2, with [.sup.14C]CT; for lane 3, with the vehicle solvent, which is the same as that used for assays 1 to 5; for lane 4, with [14C]CE and 0.1 M of the aminoalkyl sterol; and for lane 5, with [.sup.14C]CE and a concentration of 1 M of the aminoalkyl sterol.

[0211] It is observed that the presence of the aminoalkyl sterol causes an inhibition of ChEH; this inhibition is dependent on the concentration of aminoalkyl sterol.

[0212] Moreover, a study of this inhibition on homogenate was also carried out. The protocol is as follows: the MCF7 cells are detached with trypsin and taken up with RPMI medium containing 5% FBS. The cell suspension obtained (60 million cells) is centrifuged, washed with cold PBS and resuspended in 1 ml of 20 Tris-HCl buffer (pH=7.4; 150 mM KCl). The cells are lyzed by means of five freezing/thawing (liquid nitrogen/37 C.) cycles. The solution is centrifuged at 1200 rpm at 4 C. for 10 minutes. The supernatant is recovered and the amount of proteins is determined by the Bradford method. The measurement of the ChEH activity on MCF7 cell lysate is carried out as follows: the enzymatic activity is measured on 150 g of proteins in a final volume of 150 l containing 125 l of ChEH buffer (Tris-HCl, pH 7.4, 150 mM KCl) and 15 l of MCF7 proteins. The IC50 values were compared and it was noted that (IC50).sub.cells=0.6 M, whereas (IC50).sub.homogenate=11.2 M.

[0213] This difference between the IC50 values shows that the aminoalkyl sterol tested exhibits properties of preventing the occurrence of cancers.

[0214] In addition, for the DDA compound, results analogous to those present in example 6 for PBPE were determined. The protocol used is strictly identical to that which was described in detail in example 5 for the treatment with tamoxifen, with the one difference that the daily intratumor injections are carried out at a concentration of 10 M for injection volumes of 100 l. The results are collated in the table below:

TABLE-US-00009 Day 28 Day 37 Tumor size OCDO Tumor size OCDO (% (% (% (% Molecule control) control) control) control) Nontreated 100 100 100 100 control DDA 65.4 7 14.5 3 28.3 8 4.5 3

[0215] It was therefore established in this example that, firstly, the DDA product inhibits the OCDO and, secondly, in vivo, it reduces the tumor size.

EXAMPLE 11: INHIBITION OF OCDO BY INTRACELLULAR CHOLESTEROL TRANSPORT MODULATORS AND ARYL HYDROCARBON RECEPTOR (AHR RECEPTOR) MODULATORS

[0216] Two intracellular cholesterol transport modulators were tested, namely progesterone and U18666A (3--(2,20-(diethylamino)ethoxy)androst-5-en-17-one) (see Liscum L et al., J. Biol. Chem., vol. 270 (26) p. 15443-15446, 1995) (lanes 2 and 3, respectively).

[0217] Two Ahr receptor (aryl hydrocarbon receptor) modulators, namely 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(A)pyrene, were also tested (see lanes 4 and 5, respectively). Finally, two Ahr receptor antagonists were tested, namely resveratrol and 1,3-dichloro-5-[(1E)-2-(4-chorophenyl)ethenyl]-5-benzene (PDM2) (see: Casper R. F. et al., Mol. Pharmacol., 1999 October; 56(4); 784-90 and de Medina et al., J. Med Chem., 2005 Jan. 13; 48(1): 287-91).

[0218] The following experiments were carried out: MCF7 tumor cells were placed in the presence of 0.5 M of [.sup.14C]CT and were then incubated for 24 hours in the presence of one of the following products: [0219] 1. vehicle solvent (0.1% ethanol in a PBS buffer) serving as a control (lane 1) [0220] 2. 10 M of progesterone [0221] 3. 10 M of U18666A [0222] 4. 100 nM of TCDD [0223] 5. 10 M of benzo(A)pyrene [0224] 6. 10 M of resveratrol [0225] 7. 10 M of PDM2 (Ant. 1).

[0226] Thin-layer chromatographies were carried out and FIG. 24 represents the plate obtained. The OCDO production in the treated cells was quantified from the spots of the various lanes, by applying the protocol given in detail in example 7. The results of this quantification are represented by the histogram of FIG. 24.

[0227] The amounts of OCDO produced by MCF7 cells, when they are incubated according to the same protocol as defined above with other Ahr receptor antagonists, was also measured, the control test being brought to 100 and the OCDO production being expressed as % of the control (same control as above). The results are given in the following table:

TABLE-US-00010 Molecule OCDO production (concentration by MCF-7 10 M) (% control) Control 100 Ahr receptor Resveratrol <1 antagonist Ant 1 (10) <1 Ant 2 (10) <1 Ant 3 (10) <1 Ant 4 (10) <1 Ant 5 (10) <1 Ant. 1: (E)-1-(4-chlorophenyl)-2-(3,5-dichlorophenyl)-ethene; Ant. 2: (E)-1-(4-methoxyphenyl)-2-(3,5-fluorophenyl)-ethene; Ant. 3: (E)-1-(4-fluorophenyl)-2-(3,5-fluorophenyl)-ethene; Ant. 4: (E)-1-(4-trifluoromethylphenyl)-2-(3,5-trifluoromethylphenyl)ethene; Ant. 5: (E)-1-(4-fluorophenyl)-2-(3,5-dimethoxy-phenyl)ethene

[0228] It therefore appears that the intracellular cholesterol transport modulators and the Ahr receptor antagonists can inhibit OCDO formation and can consequently be used for their anticancer effect.