MXPA99008068A - Method of suppressing tumor growth with combinations of isoprenoids and statins - Google Patents

Abstract

A methodof inhibiting the growth of tumor cells is disclosed. In one embodiment, this method comprises the step of exposing tumor cells to an effective amount of a composition comprising at least two compounds selected from the group consisting of tocotrienols, statins and ionones.

Description

METHOD OF SUPPRESSION OF TUMOR GROWTH WITH COMBINATIONS OF ISOPRENOIDS AND STATINES BACKGROUND OF THE INVENTION Several constituents derived from mevalonate (isoprenoid) of fruit, vegetables and cereals suppress chemically initiated carcinogenesis. This action has been attributed to the induction mediated by isoprenoids of detoxifying activities and to the antioxidant activity of some isoprenoids. None of these actions explains the powerful impact that isoprenoids have in the stage of promotion / advance the chemically initiated carcinogenesis and on the growth of established and chemically implanted tumors (reviewed by Elson, 1995, Elson and Yu, 1994). Isoprenoids differ substantially in terms of the impact they have on tumor growth. Isoprenoids suppress through post-transcriptional actions (Correll, et al., 1994, Parker, et al., 1993, DM Peffley and AK Gayen, personal communication), 3-hydroxy-3-methylglutaryl coenzyme A activity (HMG) -CoA) reductase, the activity considered as limiting the speed of cholesterol synthesis. Statins are competitive inhibitors of HMG-CoA reductase. Correlations between the end-stage tumor suppression potency of several isoprenoids and their impact on HMG-CoA reductase activity are close to unity. Tumor reductase activity differs from liver reductase activity by its resistance to sterol feedback regulation. Tumor activity, however, retains a high sensitivity to post-transcriptional regulation caused by several isoprenoids. As a consequence of the isoprenoid-mediated suppression of HMG-CoA reductase activity, the intermediate sets of the mevalonate pathway become limiting for the post-translational processing of proteins associated with growth (reviewed by Elson, 1995; Elson and Yu 1994). A recent review presented a list of structurally diverse isoprenoids with a variable ability to suppress mevalonate synthesis, (Elson, 1995). COMPENDIUM OF THE INVENTION In one embodiment, the present invention is a method for inhibiting the growth of tumor cells. The method comprises treating the cell with a combination of at least two products of the mevalonate pathway selected from the group consisting of statins, ionones and tocotrienols. In a preferred embodiment, the ionone is selected from the group consisting of beta-ionone; 6-10-dimethyl-undec-3, 5-ene-2, 9-dione; 6, 10-dimethyl-9,10-epoxy-undec-3,5-ene-2-one; 9,10-diacetoxy-6,10-dimethyl-undec-3,5-ene-2-one; 6, 10-dimethyl-9, 10-diol-undec-3,5-ene-2-one and alpha-ionone. Preferred tocotrienols include d-gamma-tocotrienol, 2-ethylethocotrienol, d-delta-tocotrienol and d-tocotrienol. Preferred statins include lovastatin, pravastatin, simvastatin and fluvastatin. In another form, the present invention is a pharmaceutical composition for the treatment or prevention of tumors, comprising effective amounts of at least 2 agents selected from the group consisting of tocotrienols, statins and ionones. It is an object of the present invention to prevent or reduce tumor growth and metastasis. It is another object of the present invention to increase the survival duration of a tumor patient after tumor detection. It is another object of the present invention to prevent the formation of tumors. Other objects, advantages and features of the present invention will be apparent to one skilled in the art after review of the specification, claims and drawings. DESCRIPTION OF THE DRAWINGS Figure 1 graphically represents the dose-dependent impact of knockers on the proliferation of B16 melanoma cells. Figures 2 (A) and (B) illustrate the effects of combinations of gamma-tocotrienol, beta-ionone and carvacrol on melanoma B 16 cell populations. Figure 2 (A) illustrates the effects of gamma-tocotrienol and beta -ionona. Figure 2 (B) illustrates the effects of carvacrol and beta-ionone. Figure 3 is a survival curve for host mice receiving diets enriched with isoprenoid after detection of a solid implanted B 16 melanoma. DESCRIPTION OF THE INVENTION In a modality, the present invention is a method for inhibiting the growth of tumor cells by exposure of tumor cells to a combination of isoprenoids. In one embodiment of the present invention, the combination comprises at least one tocotrienol and at least one ionone. In another embodiment of the present invention, the combination comprises at least one tocotrienol and at least one statin. In a third embodiment of the present invention, the combination comprises at least one statin and at least one ionone. We contemplate that another suitable embodiment of the present invention would be a combination comprising these three products of the mevalonate pathway. By "tocotrienol", we understand a member of the following group: the family of vitamin E consists of a mixture of vitamors, which consist in general terms of tocopherols and tocotrienols. Tocotrienols are epimers of the corresponding tocopherols. The following list describes representative tocopherols and tocotrienols. d-alpha-tocopherol: 2, 5, 7, 8-tetramethyl-2- (4,8,13-trimethyltridecyl) -chroman-6-ol d-alpha-tocotrienol: 2,5,7,8-tetramethyl-2 - (4, 8, 12-trimethyltrideca-3, 7, 11-trienyl) -chroman-β-ol d-beta-tocopherol: 2,5,8-tetramethyl-2- (4,8,13-trimethyltridecyl) - Chroman-6-ol d-beta-tocotrienol: 2, 5, 8-trimethyl-2- (4, 8, 12-trimethyltrideca-3, 7, 11-trienyl) -chroman-6-ol d-gamma-tocopherol: 2, 7, 8-tetramethyl-2- (4,8,13-trimethyltridecyl) -chroman-6-ol d-gamma-tocotrienol: 2,7,7-trimethyl-2- (4,8,13-trimethyltrideca) 3, 7, 11-trienyl) -chroman-6-ol d-delta-tocopherol: 2,8-dimethyl-2- (4,8,13-trimethyltridecyl) -chroman-8-ol d-delta-tocotrienol: 2 , 8-dimeti1-2- (4,8, 12-trimetiItrideca-3,7, 11-trienyl) -chroman-6-ol d-tocopherol: 2-methyl-2- (4, 8, 12-trimethyltridecyl) - Chroman-8-ol d-tocotrienol: 2-methyl-2- (4, 8, 12-trimethyltrideca-3, 7, 11-trienyl) -chroman-6-ol 2-demethyl-trichlorienol: 2- (4, 8, 12 -trimethyltrideca-3, 7, 11-trienyl) -chroman-6-ol the preferred tocotrienols include d-gamma-tocot rienol and 2-demethylocotrienol. Other preferred tocotrienols include d-beta-tocotrienol, d-delta-tocotrienol and d-tocotrienol. By "ionone" we mean a member of the following group: ionones are compounds related to carotenoids widely distributed in nature. Alpha-ionone and beta-ionone and several oxygenated derivatives are widely present in plants in free and conjugated forms. Biological activities seem restricted to functions such as phytoalexins. Ionones are also formed by the oxidation of carotenoids thermally and photochemically mediated. Preferred ionones include beta-ionone (4-2,6,6-trimethyl-1-cyclohexen-1-yl) -3-buten-2-one; 6-10-dimethyl-undec-3, 5-ene-2, 9-dione; 6-10-dimethyl-9, 10-epoxy-undec-3,5-ene-2-one; 9,10-diacetoxy-6,10-dimethyl-undec-3,5-ene-2-one; and 6, lO-dimethyl-9,10-diol-undec-3,5-ene-one. Other preferred ionones include alpha-ionone (4- (2,6,6-trimethyl-2-cyclohexen-1-yl) -3-buten-2-one.) By "statin", we mean one member of the following group: statins are derivatives of fungal metabolites (ML-236B) compactin / monocalin K) isolated from Pythium ultimum, Monacus ruber, Penicillium citrinum, Penicillium brevicompactum and Aspergillus terreus. These 3-hydroxy-3-3-methyl glutaric acid (HMG) analogues compete with HMG-CoA for binding site on substrate in HMG-CoA reductase. Statins are available by prescription in the United States of America. For example lovastatin (Mevacor / Merck), simvastatin (Zocor / Merck), pravastatin (Pravachol / Bristol-Myers Squibb) and fluvastatin (Lescol / Sandoz). There are several other statins in clinical research, including a statin in late-stage trials at Warner-Lambert. The more lipophilic statins have been associated with certain skeletal muscle disorders (myositis, rhabdomyolysis), but most of the side effects reported in clinical trials have been mild and tolerable (headache, abdominal pain, constipation, flatulence and diarrhea). (Pedersen, T.R., N. Engl. H. Med. 333: 1350-1351, 1995; Kobashigawa, J.A., et al., N. Engl. J. Med. 333: 621-627, 1995). Following are some of the preferred statins. Derivatives Trade name Normal Dose Range Dosage form (mg / d) (mg / d) lovastatin Mevacor 10-80 20-40 pravastatin Pravachol 10-40 20-40 simvastatin Zocor 5-40 5-10 Synthetic compound Fluvastatin Lescol 20-80 20-40 The following list describes the chemical formulas of the preferred statins.

Lovastatin: (ÍS (la (R), 3alpha, 7beta, 8beta (2S, 4S), dabeta)) -1,2,3,7,8, 8a-hexahydro-3,7-dimethyl-8 (2- ( tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) ethyl) -l-maftalenyl-2-methylbutanoate Pravastatin sodium: 1-Naphthalene-heptonoxy, 1,2,6,7,8a-hexahydro- beta, delta, 6-trihydroxy-2, methyl-8 (2-methyl-l-oxybutoxy) -1, monosodium salt (lS-dalfa (betas, deltas), 2alpha, 6alpha, 8beta (R), 8a alpha Simvastatin: butanoic acid, ester (1S-lalfa, 3alpha, 7beta, 8beta, (2S, 4S), 8abeta of 2,2-dimethyl, 1,2,3,7,8, 8a-hexahydro, 3, 7-dimethyl-8 - (2-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl) ethyl) -1-naphthalene Fluvastatin sodium: acid (R, S- (E) - (+/-) -7- (3 ( 4-fluorophenyl) 1- (1-methylethyl) -lH-indol-2-yl) -3,5-dihydroxy-beta-heotenoic monosodium salt Preferred statins include lovastatin, pravastatin, simvastatin and fluvastatin.The following examples demonstrate a synergistic effect when B16 melanoma cells are treated with combinations of tocotrienols , lovastatin and ionone. In an example below, a 68% reduction in the number of B 16 melanoma cells (measured after 48 hours) was obtained. This represented a synergistic effect of 9% compared to the additive sums of the individual effects. In in vivo tests in which the survival after implantation of mice carrying melanoma was measured, the P values for the three comparisons of isoprenoid treatments versus combined treatments fall within the range of 0.66 - 0.16 while the P values for the three Isoprenoid comparisons versus isoprenoid treatments are greater than 0.88. we observed first that all treatments significantly increased the duration of survival (P <; 0.03). Differences between single treatment effects were not significant. The nonparametric test P values fall within the range of 0.64 and 0.95. when we tested the significance of the differences between single treatment and mixing effects, nonparametric P values fell within a range of 0.16 to 0.64. the trend towards values lower than P suggests a possible synergy. By "synergistic" we refer to a percentage reduction in the number of cells of at least 5% additional compared to the additive sum of the individual effects or an increase in the host survival capacity of 5% on the additive sum of the individual effects. To achieve the present invention we evaluate the tumor suppression potency of several diverse isoprenoid compounds in vitro and the potency of two of them in vivo, d-gamma-tocotrienol and beta-ionone. We accumulate findings in the sense that isoprenoids have little in common among them, except the characteristic of sharing a common precursor, isopentenyl pyrophosphate, a suppressed proliferation of melanoma cells and show that the examples of individual isoprenoids tested in binary mixtures are additive . We also report that a relevant dietary intake of d-gamma-tocotrienol suppressed the growth of implanted tumors. The study described below in the examples estimated the structurally diverse isoprenoid concentrations required to inhibit growth in a population of murine B16 melanoma cells (FIO) during a 48-hour incubation by 50% (IC50 value). The IC 50 values for d-limonene and perilylic alcohol, the monoterpenes in phase I test, were respectively 450 and 250 μmol / l; the related cyclic monoterpenes (perilaldehyde, carvacrol, thymol), an acyclic monoterpene (geraniol) and the final ring analog of beta-carotene (beta-ionone) had IC 50 values within a range of 120 to 150 μmol / L. The IC50 value estimated for farnesol, the side chain analogue of the tocotrienols (50μmol / L) was found halfway between the alpha-tocotrienol value (HOμmol / L) and the estimated values for gamma-tocotrienol (20μmol / L) and delta-tocotrienol (10 mmol / L). A novel tocotrienol that does not have methyl groups in the tocol ring proved to be extremely potent (ICso ^ 0.9μmol / L). In the first of two diet studies, experimental diets were provided to female C57BL mice weaned for 10 days before and for 28 days after implantation of B16 melanoma (FIO) of aggressive and highly metastatic growth. The isomolar substitution (116μmol / kg of diet) and the equivalent of vitamin E (928μmmol / kg of diet) of d-gamma-tocotrienol by dl-alpha-tocopherol in the diet of AIN-76a produced delays of 36% and 50% , respectively, in tumor growth (P <0.05). In the second study, melanomas were established before the mice received experimental diets formulated with 2 mmol / kg of d-gamma-tocotrienol, beta-ionone individually and in combination. A fourth diet with 4 mmol / kg d-gamma-totrienol was formulated (figure 3). Each treatment increased (P <0.03) the duration of host survival. The present invention stems from a finding in the sense that the effects of some individual isoprenoids were additive and the effects of other individual isoprenoids were synergistic. This finding suggests that a particular combination of isoprenoids would be suitable for chemotherapeutic applications. The methods of the present invention can be carried out in various modalities. In one embodiment, the human or animal subject receives the combination of isoprenoids in a pharmaceutical or veterinary composition that contains a safe and effective dose. In another embodiment, the subject receives a food that has been enriched with the isoprenoid combination. Statins are now available only by prescription and preferably are not added to foods. Foods for humans and animals as well as pharmaceutical preparations for use in the methods of the present invention are those that contain the combination of selected isoprenoids combined in addition with conventional animal feeds, food supplements for humans or approved pharmaceutical excipients and diluents . Highly active tocotrienols include d-gamma-tocotrienol, d-delta-tocotrienol and 2-demethyltocotrienol, both occurring naturally. D-gamma-tocotrienol and d-delta-tocotrienol can be extracted from fractions rich in tocotrienol from rice bran oil or palm oil by published methods. 2-Demethyltocotrienol can be extracted from fractions rich in tocotrienol from rice bran oil by published methods. Beta-ionone can be obtained from several commercial sources. For example, beta-ionone can be found in catalog number W25950-0, Aldrich Flavors and Fragrances; beta-ionone is also listed in the catalogs of Aldrich Fine Chemical and Sigma. Other ionones appear in lists in the aforementioned catalogs and in Bedoukian Research Distinctive Perfume & Flavor Ingredients Lovastatin can be obtained very easily in commerce. The combination of isoprenoids, in addition to being added to the patient's food, may also be administered in the form of pharmaceutical or veterinary compositions such as for example enteric coated tablets, capsules, powders, solutions or emulsions. The precise amount to be administered will depend on the isoprenoids used, the route of administration, the patient's weight and the nature of the condition. We observed that the melanoma cell line used for the following examples is extremely resistant to chemotherapeutic measures. Therefore, the actual dosage used for the treatment of patients may be lower than the dosage proposed from our experimental model. In general terms, the amounts of the agents required in a pharmaceutical combination will provide a daily intake substantially lower than the quantities required when the agents are administered individually. Perilyl alcohol, an isoprenoid in clinical evaluation to determine its efficacy as an antitumor agent, is administered at levels above 8 g / day; lovastatin is clinically effective, but poorly tolerated, for the treatment of brain tumors in doses of -2 g / d (more than 20 times the dose administered for hypercholestero- memia). Tocotrienols effectively decrease cholesterol levels when consumed at dose levels of less than 300 mg / day. Due to the synergistic action of the agents, we anticipate a preferable mixture that will contain 250 mg of tocotrienol / 2000 mg of ionone / 150 mg of statin (-2mg / kg of body weight) per day. A maximum preferred dosage will contain 500 mg of tocotrienol / 4000 mg of ionone / 300 mg of statin per day. A representative pharmaceutical enteric coated tablet has the following formula: Active ingredients: d-gamma-tocotrienol 50 mg 6.10-dimethyl-9, 0-epoxy-undec-3, 5-ene-2-one 400 mg statin 30 mg Excipients / fillers Microcrystalline cellulose Sodium starch glycolate Corn starch Hydrogenated vegetable oil wax Talcium magnesium stearate The preferred normal dosage per day is 5 tablets. A bar or cookie can be of any desired type prepared first and partially dosed with a small amount of liquid containing the active ingredients: d-gamma-tocotrieno 150 mg 6, 10-dimethyl-9, 0-epoxy-undec-3, 5-ene-2-one 400 mg It is preferable that suitable flavors be present in the bar or biscuit to provide an acceptable taste. It is essential that the active ingredients are added after any baking or heating process and the cooling determined since the tocotrienols do not resist heat. A bar or cookie of this type can be packaged advantageously in an airtight package. Since tocotrienols and ionones are relatively non-toxic, higher doses than the highest number, particularly with the composition, enteric coating may be administered if desired. Higher doses may, however, markedly reduce the intestinal flora and cause gastrointestinal disorders. It is recommended that, when administering the compositions of the present invention, the daily intake of vitamin I is reduced. The more lipophilic statins (with propensity for fats) have been associated with some skeletal muscle disorders (myositis, rhabdocolysis), but most of the side effects reported in clinical trials have been mild and tolerated (headache, abdominal pain, constipation). , flatulence and diarrhea). The patients (Thibault, et al., Clinical Cancer Research 2: 483-491, 1996) had intakes of almost 2 g / day (25 mg / kg body weight). It will be apparent to those skilled in the art that various modifications or changes may be made without departing from the spirit or scope of the present invention. Thus, the invention is only limited by the appended claims. Examples Abbreviations used: HMG CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IC50, the concentration required to suppress the increase in the melanoma cell population by 50%; TRF, fraction rich in tocotrienol of palm oil; TRF25, fraction rich in orcotol free tocotrienol of rice bran oil. A. Materials and Isoprenoid Methods: d-limonene (97%), perilylic alcohol (99%), perilaldehyde (92%), carvacrol (98%), thymol (98%), beta-ionone (96%), geraniol ( 98%), farnesol (96%) and dl-alpha-tocopherol (97%) were purchased from Aldrich Chemical, Milwaukee, Wl. An abbreviated list of concentrated natural sources and aromas characteristic of these isoprenoids is shown below in Table 1. A preliminary study revealed the very potent antitumor action of the fraction rich in tocotrienol, free of oryzanol from rice bran oil (TRF25) prepared by molecular distillation (Dr. Laxman Singh, Vitamin, Inc., Chicago, IL). The fraction consisted of 6% of d-alpha-tocopherol, 12.5% of d-alpha-tocotrienol, 21% of d-gamma-tocotrienol, 10% of d-delta-tocotrienol, 4.5% of d-tocotrienol, 17% of d-2-demethyl tocotrienol, 18% of unidentified isomers of tocotrienol and 10% of sterols and triglycerides (Qureshi, et al., unpublished data). The major constituents, d-alpha-tocotrienol, d-gamma-tocotrienol, d-delta-tocotrienol, and d-2-demethyl tocotrienol were isolated by Advanced Medical Research Madison, Wl. A chromatographic procedure was developed to separate d-gamma-tocotrienol from the tocotrienol-rich fraction (TRF) of palm oil (36% of d-gamma-tocotrienol, 18% of d-alpha-tocotrienol, 12% of d-delta -tocotrienol and 22% of d-alpha-tocopherol), a gift from the Palm Oil Research Institute of Malaysia, Kuala Lumpur, Malaysia. Silica gel (Merck, ßOμ, 150 g) was suspended in hexane in a 350 ml glass funnel with a frit disk. The gel was washed with 1 L hexane before being charged with 5 g of the TRF in 20 ml of hexane. The touches were eluted with the sequential applications of 500 ml of diethyl ether mixtures (5%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 25% and 30%) in hexane. The elusion of each solvent application in a filter bottle was accelerated by the application of vacuum produced by water aspiration. The eluted products were dried in vacuo, the residues dissolved again in hexane and identified in accordance with the retention time and the absorption profile using an analytical HPLC system. The fraction eluted with diethyl ether 18% was predominantly d-gamma-tocotrienol (98%). Determinations of ICs0: murine B16 melanoma cells (FIO), a line of tumor cells with high metastatic potential (Tsukamoto, et al., 1991) obtained from Dr. William B. Ershler, were cultured in monolayer culture (flasks 35 x 10 mm) in 3 ml of medium RMP1 1640 (Sigma) supplemented with 10% of newborn calf serum (GIBCOBRL, Grand Island, NY) and 80 mg / L of gentamicin (Sigma, St. Louis, MO). The cultures, seeded with 1-I.5xl05 cells were incubated for 24 hours at 37 ° C in a humidified atmosphere of 5% C02. The isoprenoids, dissolved in absolute ethanol, were added at 24 hours (time 0); all cultures contained 5 ml of ethanol / L (85μmol / L). The cultures were incubated for an additional 48 hours. The media was removed and the monolayers were washed twice with a balanced salt solution of Hanks (Sigma) and then incubated with a solution of Trypsin-EDTA (Sigma) at a temperature of 37 ° C for two minutes. Trypsin was deactivated by suspending the cells in a medium containing 10% fetal bovine serum (Sigma). The cells were formed into pellets at 250 x g and resuspended in a balanced salt solution of Hanks. Viable cells, cells that excluded triptan blue 0.4% (GIBCOBRL). They were counted with a hemocytometer; cells counted at 24 hours of final cell counts were counted to provide a Estimation of the net increase in the number of cells. The calculation of the concentration of an isoprenoid required to inhibit the net increase in cell count at 48 hours by 50% (IC50) is based on graphs of data from 3 or more evaluations. Animal studies: this evaluation of the antitumor potency of several isoprenoids was extended with 2 diet studies. The first study examined the impact of the relevant ingestion of d-gamma-tocotrienol on the growth of B16 melanomas (FIO) in host mice. The second study evaluates the impact of pharmacological intakes of d-gamma-tocotrienol and beta-ionone on survival after implantation of mice carrying melanoma. We prepare a basal dietary mixture first, defined according to the formulation AIN-76A (American Institute of Nutrition, 1977) but free of corn oil and dl-alpha-tocopherol. These ingredients and corn oil without vitamin E were purchased from Teklad Test Diets, Madison, Wl. Stock solutions of dl-alpha-tocopherol (80μ mol / g) and d-gamma-tocotrienol (80μmol / g) were prepared with corn oil without vitamin E. Stock solutions of the blubber, diluted with corn oil without vitamin E were mixed with the basal diet in order to offer finished diets containing 5% corn oil and the specified concentrations of tocol. Where mentioned, beta-ionone was added to the oil. The diets, mixed weekly, were stored under refrigeration. Feeding rates were cleaned and refilled daily. Experiment 1: Wean female C57BL mice (Harlan-Sprague Dawley, Madison, Wl) were housed in groups of 4 in wood particles in plastic cages and the temperature was maintained at 25 ° C with a light-dark filter of 12 hours. The four groups of mice (20 / group) were fed with experimental diets for 10 days before and for 28 days after the implantation of B16 melanoma cells. The divided graphic design consisted of 4 treatments comprising 2 knobs, dl-alpha-tocopherol and d-gamma-tocotrienol, each presented in two levels, a diet of 116 and 924μmol / kg. This design allowed two comparisons of equal diets in tocol content and a comparison of approximately equal diets in equivalents of d-alpha-tocopherol. Since we did not have a definitive estimate of the biological activity of d-gamma-tocotrienol, we consider reports in the sense that 2,5,8 (d-beta-) and 2,7,8 (d-gamma-) trimethyl Tocopherols have a similar oxygen removal activity (maximum 66% of the activity of 2,5,7,8 (d-alpha-) tetramethyl tocopherol) and as a class, tocotrienols have from 3 to 30% of the biological activity of the tocopherols (Karnal-Eldir and Appelqvist, 1996) to develop the appropriate estimate that d-alpha-tocopherol has minimally, 6 times the biological activity of d-gamma-tocotrienol. We corrected later to take into account the biological activity of the mixture of dl-alpha-tocopherol (70% of the activity of d-alpha-tocopherol) to reach the 8: 1 ratio between tocotrienol and tocopherol used in the formulation of experimental diets. Melanoma cells, cultured and harvested according to the previously described (Shoff, et al., 1991), were washed twice with RPMI 1640 containing 20% fetal bovine serum (GIBCOBRL). The cells formed in pellets were suspended in RPMI 1640 and counted (98% viable cells) after a 1:20 dilution in 0.4% triptan blue. The cells were then diluted in RPMI 1640 (1 x 108 cells / 1) and 0.1 ml of the suspension (1 x 104 cells) was injected subcutaneously into the flank of each mouse. The study ended on day 28 when the first mouse died, a mouse in the control group. The mice were sacrificed by CO 2 overdose and the tumors were removed and weighed. In the College of Agricultural and Life Sciences Animal Care Co mittee (Animal Care Committee of the Faculty of Agriculture and Life Sciences) reviewed and approved the protocol.

Experiment 2: weaned C57BL female mice (n = 60, Harian Sprague-Dawley) were acclimated to the housing conditions and were given an AIN-76A diet (116 μmol dl-alpha-tocopherol / kg diet) in accordance with described above. Tumors were implanted and the mice continued to receive the AIN76A diet. Starting on day 8 after implantation, the mice were palpated daily to determine the presence of a tumor. Tumors were first detected on day 14. Random numbers were generated to assign each mouse in a sequential subset of 5 to a diet. Experimental treatments provided 2 and 4 μmol of d-gamma-tocotrienol / kg of diet. We additionally tested the impact of beta-ionone (2 μmol of beta-ionone / kg of diet AIN-76A) and the impact of a mixture of the two isoprenoids (2 mmoles each / kg of diet) on the survival of the mice. The mice continued with their respective experimental diets; the moribund mice, identified by the supervisor trained in research on study animals without knowledge of the experimental design, were sacrificed over C02 doses and the tumors were removed and weighed. The protocol was reviewed and approved by the Animal Care Committee of the Faculty of Agriculture and Life Sciences. Statistical methods: StatView and SuperANOVA (Abacus Concepts, Berkeley, CA) were used for the evaluation of the effects mediated by the treatment. The differences mediated by treatment in terms of body weight and tumor weight, days until the appearance of the tumor and days until morbidity were identified with analysis of divided graph of variance and t-tests paired with the mean of least squares. Differences mediated by treatment in days to morbidity were also evaluated using parametric tests (paired t tests) and nonparametric tests (Wilcoxon's indicated range) (Haycock, et al., 1992). B. Results Figure 1 is a representative evaluation of the dose-dependent impact of 5 different knockers on the proliferation of melanoma B16 cells. Cultures (3 L) seeded with 1-1.5 x 105 cells were incubated for 24 hours before the introduction of the tocolles. Viable cells were counted at 48 hours after the addition of the tocolles. The cell count at 24 hours (zero time) is illustrated by a dashed line. The intersection of the horizontal solid line and the line marked for each tocol indicates the concentration at which the tocol suppressed by 50% increase in the number of cells during the incubation (IC 50 value) • The values of IC > or (mean, standard deviation and n) for all the isoprenoids tested are shown in table 1. With reference to figure 1, dl-alpha-tocopherol had no impact on the number of cells. The growth suppression potency of the individual tocotrienols was inverse to the number of methyl groups in the 6-chromanol ring: d-alpha-tocotrienol (methyl groups in the carbons 5,7,8 and 2) < < d-gamma-tocotrienol (methyl groups in carbons 7,8 and 2) < d-delta-tocotrienol (methyl groups in carbons 8 and 2) «d-2-demethyl tocotrienol (non-methyl groups) (table 1). Within this series of tocotrienols, the IC5o value was inversely related to the number of methyl groups in the 6-chromanol ring. Similarly, the IC50 values calculated from the graphs for the most polar monotertenoid alcohols, perilylic alcohol ((R) 4-isopropenyl-1-methanol-cyclohexane), perilaldehyde ((R) -4-isopropenyl-1- carboxaldehyde cyclohexene), thymol (5-methyl-2-isopropylphenol), carvacrol (5-isopropyl-2-methylphenol) and geraniol (trans 3,7-dimethyl-2,6-octadien-l-01) were much lower than Values for d-limonene ((R) 4-isopropenyl-l-methyl-1-cyclohexene). The IC50 value for beta-ionone, the beta-carotene extreme ring analog, corresponded to the value of the most polar monoterpenes (Table 1). The IC 50 for farnesol (trans, trans 3, 7, 11-trimethyl-2, 6, 10 dodecatrien-1-ol), a sesquiterpene and a structural analog of the side chain of the tocotrienols, fell by half between the IC 50 of d-alpha-tocotrienol and d-gamma- and d-delta-tocotrienol.

The first study of the diet evaluated the impact of d-gamma-tocotrienol and dl-alpha-tocopherol on the days until the detection of a solid tumor and the growth of implanted B 16 melanomas. As noted above, the design allowed for two comparisons of treatments that provided an equal tocol concentration (diet with 116 and 928μmol / kg) and a comparison of treatments that provided a diet of approximately 80umol of d-alpha-tocopherol equivalents (35 mg) / kg. At the time of tumor implantation, the body weight of mice that received the diet high in tocopherol was significantly lower than the body weight of mice that received the low tocol diets (Table 2). At 28 days after implantation, the melanomas represented 15% of the weight of the mice that received an AIN-76 A diet. Two tocoles were tested, each in two levels. The analysis of the divided plot of variance showed that the effects of tocols (P <0.01) and levels (P <0.04) on the tumor weight for day 28 were significant; no evidence of an interaction between the two factors was observed (P <0.77). the analysis of least squares confirmed the antitumor action of gamma-tocotrienol (table 2). The other measurement of tumor growth, days after the time until tumor detection showed that tocol (P <0.03) level (P <0.01) and the interaction (P <0.02) were significant. The least squares analysis showed that the effect of the treatment provided by a diet of 928 μmol of d-gamma-tocotrienol / kg on this measurement of tumor growth was significantly different from the effects of the other treatments. We then determined that the effects of isoprenoids on the growth of B16 melanoma cells in culture are additive, and in some cases synergistic. Figure 2 (A) illustrates the additive effects of gamma-tocotrienol and beta-ionone on populations of B16 melanoma cells. The values are means +/- standard deviation, n = 20; SEM combined = 36 (xlO4). Cell counts calculated to show the population decreased in relation to the control appear in the list in the inserted table. Figure 2 (B) illustrates the additive effects of carvacrol and beta-ionone on B16 melanoma cell populations. The values are means +/- standard deviation, n = 28; SEM combined = 62 (xlO4). The cell counts calculated to show the population decreased in relation to the control appear in the list in the inserted table. a_h Stocks that do not share a superindices (a > b> c> d> g> g) are different (P < 0.001). With reference to Figure 2, B16 melanoma cells were incubated with beta-ionone and d-gamma-tocotrienol in one test (Figure 2 (A)). D-gamma-tocotrienol (7.5μ mmol / l) and beta-ionone (50μmol / 1) reduced cell counts at 48 hours in 19% and 10%, respectively (Figure 2 (A)). The 34% reduction in the number of cells reached with the isoprenoids in pairs exceeded the 29% reduction predicted by the sum of the individual effects (Figure 2 (A), insert). At higher concentrations, d-gamma-tocotrienol (15μmol / L) and beta-ionone (100μmol / L) reduced cell counts at 48 hours in 34% and 16%, respectively; the additive effect, a reduction of 68% in the number of cells, was also greater than the sum of the individual effects (59%) (Figure 2 (A), insert). While the above results indicate a synergistic action of d-gamma-tocotrienol and beta-ionone, the paired study of carvacrol and beta-ionone revealed only an additive effect (Figure 2 (B)). Carvacrol (50μmol / l) and beta-ionone (75μmol / l) reduced the cell count at 48 hours in 25% and 31%, respectively; the additive effect, a 48% reduction in the number of cells, was less than the 56% reduction predicted by the sum of the individual effects. Carvacrol (100μmol / l) and beta-ionone (150μmol / l) reduced cell counts by 35% and 65% respectively; the additive effect, an 84% reduction in the number of cells, was again less than the 100% reduction predicted by the sum of the individual effects (Figure 2 (B), insert). The reduction in the number of cells predicted by the sum of the individual effects of the isoprenoids was highly correlated with the reduction achieved with the isoprenoids in pairs (r = 0.91, n = 8, P <0.01). We then asked if d-gamma-tocotrienol or beta-ionone of the diet would prolong the survival of mice with implanted melanomas. Dietary treatments were initiated after the detection of solid tumors. We also wonder if these structurally diverse isoprenoids could have an additive effect on survival. Experimental groups were constructed by randomly assigning each member of successive subsets of 5 mice to one of the experimental diets. The time to tumor detection did not vary between groups (Table 3). The treatments increased the average duration of survival by 42 +/- 4.5% and the average duration of survival by 30% (P <0.005); the differences between the duration of the survival mean of the experimental groups were not significant (table 3, figure 3). Figure 3 is a survival curve for host mice that received diets enriched with isoprenoids after detection of a solid implanted B16 melanoma. The mean survival durations appear in Table 4. The treatments with beta-ionone and gamma-tocotrienol1 yielded 2 mmol of the isobrenoid / kg of diet; the mixture provided 2 mmol of each isoprenoid / kg of diet; and gamma-tocotrienol2 provided 4 mmol of gamma-tocotrienol / kg of diet. In accordance with the experimental design, each animal within a subset of 5 was paired with another member of the subset to perform tests. The P values for the pairwise comparisons, considering normal and abnormal distributions, appear in table 4. All the analyzes revealed highly significant differences between the control and the mean of the treatment groups; Differences between the means of the control groups were not significant. What provides additional credibility to the results of in vitro analysis showing an at least additive effect of the isoprenoids is the trend illustrated in Table 4. While the P values for the three comparisons of isoprenoid versus isoprenoid treatments are greater than 0.88, the P values for the three comparisons of isoprenoid versus mixture treatments fall within the range of 0.66 to 0.16. C. Comments Melanoma B 16 (FIO) provides a rigorous model to evaluate, in vitro and in vivo, the potency of pharmacological agents (Gruber, et al., 1992, Kuwashi a, et al., 1990, Mac Neil, et al. al., 1992; Tsukamoto, et al., 1991; Shoff, et al., 1991). We now report that a diet that provides an intake of 0.4 mg of d-gamma-tocotrienol / day suppressed the growth of B16 melanoma implanted in the flank of host mice. In our most rigorous test, a diet that provided 7 μmol of d-gamma-tocotrienol or beta-ionone / day suppressed the growth of established B 16 melanomas. In vitro tests provided evidence of the additive effects of these two isoprenoids (Figure 2 (A), (B)). The diet that provides an intake of 7 μmol of d-gamma-tocotrienol / day increased the duration of survival by 35%. Whereas the fact of doubling the intake of d-gamma-tocotrienol at 14 μmol / day did not increase the survival duration (-0.21 d, P = 0.95), the addition of an intake of 7 μmol of beta-ionone to the ingestion of 7 μmol of d-gamma-tocotrienol / day showed a tendency to increase the duration of survival (+0.83 d, P = 0.65, Tables 3.4). This study evaluated the responses of established implanted melanomas to beta-ionone and d-gamma-tocotrienol, two isoprenoids with structural relationships with the two antioxidant nutrients recently discounted in relation to having tumor suppressor actions (Greenberg and Sporn, 1996). Both isoprenoids significantly increased the survival time (table 4). We observed findings in the sense that a massive intake of ascorbic acid (733μmol / d) suppressed the growth of implanted B16 melanomas (Meadows, et al., 1991). Our studies show that a diet that provides an 8-fold elevation in d-alpha-tocopherol equivalents has a marginal impact on tumor growth while a diet with d-gamma-tocotrienol provides only the equivalent of d-alpha-tocopherol. Alpha-tocopherol of the AIN-66 A diet offers a highly significant suppression of tumor growth. D. Synergistic action of lovastatin, tocotrienol and ionone i. Background Lovastatin, a fungal antibiotic, competitively inhibits 3-hydroxy-3-methylglurail coenzyme A (HMG CoA) reductase activity (J.L. Goldstein and M.S. Brown, Nature 343: 425-430, 1990). Beta-ionone (SG Yu, et al., J. Agrie, Food Chem. 42: 1493-1496, 1994), a pure isoprenoid, and related agents suppress HMG CoA reductase activity through the suppression of this synthesis ( reviewed in H.Mo. et al., Nutritional Oncology, D. Huber & amp; amp;; G. Blackburn, eds. Academic Press, New York, in print; EC. Elson, et al., Amer. Assoc. Cancer Res., In print). Tocotrienols, a group of mixed isoprenoids, suppress the reductas activity by triggering the proteolytic degradation of the enzyme (C.E. Elson, supra, in print). As a consequence of the inhibited reductase action, lovastatin and subsequent derivatives (JL Goldstein and MS Brown, supra 1990, RA Parker, et al., J. Biol. Chem. 268: 11230-11238, 1993), beta-ionone and Pure isoprenoids related (SG Yu, et al., Supra, 1994; H. Mo. et al., supra, in print), and tocotrienols (H. Mo. et al., supra, in print, RA Parker, et al., supra, 1993; AAQureshi, et al., J. Biol. Chem 261: 10544-10550, 1985; AAQureshi, et al., Proceedings of the 1996 PORIM International Palm Oil Conference, Kuala Lumpur, Malaysia, pages 168-180, 1996) lower serum cholesterol levels. Lovastatin treated cells have a multi-fold increase in HMG CoA reductase, an increase resulting in high reductase activity after inhibitor removal (J.L. Goldstein and M.S. Brown, supra, 1990). Cellular activities underlying the increase in the reductase mass include increases in transcription and mRNA processing efficiency of HMG CoA reductase and a decrease in proteolytic degradation of enzyme. Beta-ionone (SG Yu, et al., Supra, 1994) and tocotrienols (RA Parker, et al., Supra, 1993) attenuate the degree of elevation of reductase activity (SG Yu, et al., Supra, 1994) and mass (RA Parker, et al., Supra, 1993) caused by treatment with lovastatin. Mevalonate pathway intermediates are essential for the post-translational modification of proteins that play essential roles in cell proliferation (see H. Mo. et al., Supra, in print). The competitive inhibition of mevalonate synthesis imposed by statins suppresses the proliferation of cultured cells (JL Goldstein and MS Brown, supra, 1990) and the growth of implanted tumors (W. Maltese, et al., J. Clin. Invest. 76: 1748-1754, 1985; JP Jani, et al., Invasion Metastasis 13: 314-324, 1993). A 5-year follow-up of 745 hypercholesterolemic patients receiving lovastatin, up to 80 mg / day, revealed a 33% reduction in the incidence of cancers (14 observed, 21 predicted) (JA Tolbert, Arch. Intern. Med. 153 -1079-1087, 1993). A phase I trial evaluated the tolerance of lovastatin administered at progressively higher doses (from 3 to 43 cycles of 7-day courses per month) involving 88 patients with solid tumors (39 with brain tumors, 24 with prostate cancer independent of hormones). The doses were within a range of 2 to 45 mg / kg (the maximum dose for the treatment of intercholesterolemia is 80 mg / d). Sixty patients experienced a total of 128 episodes of toxicity; the incidence and severity of toxicity increased markedly when reaching a dose level of 25 mg / kg. To prevent raiotoxicity and to improve tolerance to lovastatin, ubiquinone was administered to a section of patients who received 30 mg / kg or higher doses of lovastatin. Ubiquinone did not decrease the incidence of skeletal muscle toxicity but significantly decreased its severity. The authors conclude by suggesting that high-grade gliomas represent a reasonable target for phase II trials and recommended that alternative treatment schemes aimed at achieving sustained inhibition of mevalonate synthesis be investigated (A. Thibault, et al., Clin. Cancer Res. 2: 483-491, 1996). ii. Inonone, Tocotrienol and Lovastatin act synergistically to inhibit the growth of tumor cells It is known that beta-ionone, gamma-tocotrienol and lovastatin individually suppress the proliferation of murine melanoma B16 / F10 cells. The IC 50 values for beta-ionone and gamma-tocotrienol are 140 μmol (L. He, et al., J. Nutr, 127: 668-674, 1997), and 20 μmol / l (L. He, et al., Supra). ). Using the protocol presented in He, et al., Supra, we determined that B16 cells incubated with 1.9 +/- 0.3 μmol / L of lovastatin grow at 61.4 +/- 4.6% of the speed of the controls. This value confirms the value determined using a colony formation test (J.P. Jani, et al., Supra, 1993). In vivo studies show that lovastatin (50 mg / kg) administered ip on alternate days (J.L. Goldstein and M.S. Brown, supra, 1990), beta-ionone (2 mmol / kg of diet) (L. He, et al., supra, 1997) and gamma-tocotrienol (116-2000 umol / kg of diet) individually suppress the growth of melanoma tumors B 16 murine FIO implanted. Our in vitro tests indicate a synergy between the actions of suppression of growth of the 3 agents. The action of suppression of growth of each combination is greater than the action of suppression of growth predicted by the sum of the individual effects (table 5). Table 5 presents the results of our in vitro tests. Table 5 presents a list of several concentrations of lovastatin, beta-ionone and gamma-tocotrienol and the ability to suppress growth of each treatment. Growth in treated cells is recorded as a percentage of control growth. In table 5 we see that 2 treated cells are registered as a percentage of control growth. In Table 5 we see that 2 μm of lovastatin and 150 μm of beta-ionone respectively decreased cell growth by 50% and 32% (50% and 68% of control growth) - it is predicted that the combined action of the two compounds suppresses the growth in 82% (50% + 32%) or reduces cellular growth to 18% of the control. The growth was reduced by 90% or up to 10% of the control. In a series of tests that combine lovastatin with an isoprene (tocotrienol or ionone) we made a prediction of a 66% reduction in cell count (33 +/- 5% control). However, we observed a reduction of 87% (13 +/- 4% control). Combinations of isoprenoids provided a 50% reduction in cell growth - the predicted reduction was 57% (predicted reduction of 50% of control, reduction of 43% control). It will be observed that in each combination of lovastatin, beta-ionone and tocotrienol, the combination resulted in a lower cell growth than the predicted additive value. With reference to table 5, we predict a total suppression of growth with a mixture that offers 2μm of lovastatin, 5μm of tocotrienol and 50μm of ionone. APPENDIX 1 - TABLES Table 1. The selected isoprenoid concentrations required to suppress the increase in the melanoma cell population by 50% during an incubation period of 48 hours1. A list of the representative sources and aromas characteristic of each isoprenoid is also presented. Class of isoprenoid nmol / L IC50 Monoterpenes d-limonene 3 450 +/- 43 perilylic alcohol 3 250 +/- 28 Geraniol 3 15Q +/- 19 Perilaldehyde 3 120 +/- 17 Carvacrol 3 120 +/- 15 Timol 3 120 + / -fifteen Sesquiterpenes Farnesol 2 50 B-ionone 140 +/- 23 Touched d-alpha-tocotrienol 4 110 +/- 15 d-gamma-tocotrienol 6 20 +/- 3 d-delta-tocotrienol 3 10 +/- 3 d-2-desmethyl 3 0.9 +/- 0.2 Tocotrienol dl-alpha-tocopherol > 1600 Isoprenoid class Representative source Aroma Monoterpenes d-limonene citrus peel, lemon mint perilyl alcohol citrus peel, enta, lilac sage, lavender Geraniol citrus peel, fruit Basil, rosemary Citric perilaldehyde, basil, rosemary fruit Carvacrol thyme, marjoram, mint , mint dill Thyme thyme, oregano, tangerine peel-thyme Sesquiterpenes Farnesol rose, chamomile, lavender lilac lilac B-ionone grapes, corn, apricots, wood plums Touched d-alpha-tocotrienol oils of barley, rice, oily oats, palm, olive d-gamma-tocotrienol oils of barley, rice, oily oats, palm, olive d-delta-tocotrienol oils of barley, rice, oily Oats, palm , olive d-2-demethyl bran of oily rice Tocotrienol dl-alpha-tocopherol soybean oils, oily corn wheat, barley, rice, oats, palm 1 The values are average +/- standard deviation. Table 2. Impact of d-gamma-tocotrienol on the days until the detection and growth at day 28 of B16 melanomas implanted in the flanks of mice1. Tocol - umol / kg Body weight Diet 10 d 38 d (g) (g) dl-alpha tocopherol 116 17.33 23.30 d-gamma-tocotrienol 116 17.02a 22.43 dl-alpha-tocopherol 928 16.41b 22.53 d-gamma-tocotrienol 928 b16 .83a 22.56 combined SEM 0.10 a > b a > b Tocol Tumor Liver Detection Weight Weight (d) (y) (g) dl-alpha tocopherol 19.55b 3.59a 1.18a d-gamma-tocotrienol 19.68b 2.31bc 1.12ab dl-alpha-tocopherol 19.75b 2.89ab 1.06b d-gamma -tocotrienol 22.90a 1.78c 1.03b SEM combined 0.35 0.17 0.02 a > b > c a > b a_c The means that do not share a superscript are different (P <0.05). 1 Values are means, n = 20 2 Days after implantation until the appearance of a palpable tumor. Table 3. Impact of isoprenoid-enriched diets on the survival duration of host mice with established melanomas1. Group D-gamma-tocotrienol B-ionone Mmol / kg of diet Control 0 0 d-gamma-tocotrienol 2 0 d-gamma-tocotrienol 4 0 beta-ionone 0 2 mixture 2 2 combined SEM Group Day until Days of survival Medium Median Tumor Control 15.00 13.83 ° 15.0 d-gamma-tocotrienol 14.92 18.67a 20.0 d-gamma-tocotrienol 14.92 18.46a 22.0 beta-ionone 14.92 18.27a 23.0 mixture 14.92 19.50a 20.5 combined 0.547 SEM a > b a-b Averages that do not share a superscript are different (P <0.03). 1 values are means, n = 12 Table 4. Pairwise comparisons of isoprenoid effects on the duration of survival after detection of a B16 melanoma in the flanks of mice. Paired t test Comparison of groups value t value P Control versus d-gamma-tocotrienol1 5.74 0.01 d-gamma-tocotrienol2 2.70 0.02 beta-ionone3 2.81 0.02 mixtures' 3.27 0.01 d-gamma-tocotrienol1 versus d-gamma-tocotrienol2 0.46 0.66 beta- ionone3 0.06 0.95 mixture 0.46 0.66 d-gamma-tocotrienol 'beta-ionone3 0.11 0.92 mixture4 1.36 0.21 beta-ionone-versus mixtures 4 0.93 0.37 Wilcoxon's stated range Comparison of groups value 2 P value Control versus d-gamma-tocotrienol1 .293 0.01 d-gamma-tocotrienol2 2.22 0.03 beta-ionone3 2.18 0.03 mixtures4, 2.45 0.01 d-gamma-tocotrienol1 versus d-gamma-tocotrienol 0.47 0.64 beta-ionone3 0.15 0.88 mixture 0.47 0.64 d-gamma-tocotrienol 'beta-ionone3 0.06 0.95 mixture 1.42 0.16 beta-ionone-mixtures .0.83 0.41 1 2 mmol d-gamma-tocotrienol / kg diet 2 4 mmol d-gamma-tocotrienol / kg of diet 3 2 mmol beta-ionone / kg of dita. 4 2 mmol d-gamma-tocotrienol + 2 mmol of beta-ionone / kg of diet. Table 5 Lovastatin ß-lonone? -tocotrienol growth predicted μmol / L% control 2 0 0 50 0 150 0 68 2 150 0 10 18 2 0 or 60 0 or 5 90 0 or 10 85 2 O 5 32 50 2 0 10 20 45 .5 0 0 71 3 0 or 53 0 0 10 72 0 0 20 65 .5 0 10 20 43 .5 0 20 10 36 3 0 10 0 25 3 0 20 0 18 0 50 0 90 0 100 0 84 0 0 7.5 81 0 0 15 66 0 50 7.5 66 71 0 100 7.5 64 65 0 50 15 51 56 0 100 15 32 50 0 150 0 32 0 0 15 77 0 150 15 0 APPENDIX 2 - MENTIONED LITERATURE Adany, I., et al., "Differences in sensitivity to farnesol toxicity between neoplastically - and non-neoplastically-derived cells in culture." Cancer Lett. 79: 175-179, 1994. American Institute of Nutrition. Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107: 1340-1348, 1977. Block, G., et al. , «Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence,» Nutr. Cancer 18: 1-29, 1992. 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Komiyama, K., et al., "Studies on the biological activity of tocotrienols," Chem. Pharm. Bull. 37: 1369-1371, 1989. Kuwashima, Y., et al., "Responses of a murine B16 melanoma to pharruacotherapy studied and compared with different assay systems," Cancer Res. Clin. Oncol. 116: 173-178, 1990. Laird, P.W. and Jaenisch, R., "DNA methylation and cancer," Human Molec. Gen. 3: 1487-1495, 1994. MacNeil, S., et al., "Signal transduction in murine B16 melanoma cells," Melanoma Res. 2: 197-206, 1992. Meadows, GG, et al., " Ascorbate in the treatment of experimental transplanted melanoma, »Am. J. Clin. Nutr. 54: 1284- 1291s, 1991. Meigs, TE, et al., "Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A degradation by the nonsterol mevalonate farnesol in vivo," J. Biol. Chem. 271: 7916-7922 .

Parker, RA, et al., "Tocotrienols regulate production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase," J. Biol. Chem. 268: 11230-11238, 1993. Perez -Sala, D. And Mollinedo, F., «Inhibition of isoprenoid biosynthesis induces apoptosis in human promylocytic HL-60 cells,» Biochem. Biopys. Res. Commun. 199: 1209-1215, 1994. Qureshi, A.A. , et al., "Response of hypercholesterolemic subjects to administration of tocotrienols," Lipids 30: 1171-1177, 1995.

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Claims (1)

1. CLAIMS A method for inhibiting the growth of tumor cells comprising the step of exposing tumor cells to an effective amount of a composition comprising at least two compounds selected from the group consisting of tocotrienols, statins, and ionones, wherein the amount is effective to inhibit the growth of tumor cells. The method according to claim 1, wherein the composition comprises a tocotrienol and an ionone. The method of claim 1, wherein the composition comprises a tocotrienol and a statin. The method of claim 1, wherein the composition comprises a statin and an ionone. The method of claim 1, wherein the composition comprises a statin, an ionone and a tocotrienol. The composition of claim 1, wherein the tocotrienol is selected from the group consisting of d-gamma-tocototrienol, 2-demethyltocotrienol, d-delta-tocotrienol and d-tocotrienol. The method of claim 1, wherein the tocotrienol is d-gamma-tocotrienol. The method of claim 1, wherein the ionone is selected from the group consisting of beta-ionone and alpha-ionone. 9. The method of claim 1, wherein the ionone is beta-ionone. The method of claim 1, wherein the statin is lovastatin. The method of claim 1, wherein the exposure weight comprises administering the composition as a food. The method of claim 1, wherein the exposure step comprises administering the composition in the form of a pharmaceutical agent. A method for inhibiting tumor growth comprising the step of exposing a patient to an effective amount of a composition comprising at least two compounds selected from the group consisting of tocotrienols, statins, and ionones, wherein the amount is effective for inhibit tumor growth. 14. A pharmaceutical composition comprising an effective amount of at least 2 compounds selected from the group consisting of tocotrienols, statins, and ionones, wherein the amount is effective to inhibit tumor growth. 15. The pharmaceutical composition of claim 14, comprising at least one tocotrienol and at least one ionone. 16. The pharmaceutical composition of claim 14, comprising at least one tocotrienol and at least one statin. 17. The pharmaceutical composition of claim 14, comprising at least one statin and at least one ionone. 18. The pharmaceutical composition of claim 14, comprising at least one statin, at least one ionone and at least one tocotrienol. 19. The pharmaceutical composition of claim 14, comprising beta-ionone and d-gamma-tocotrienol. The composition of claim 14, wherein the composition is part of a food. 21. The composition of claim 14, wherein the daily intake of tocotrienol is between 250 mg and 500 mg. 22. The composition of claim 14, wherein the daily ionone intake is between 2000 mg and 4000 mg. 23. The composition of claim 14, wherein the daily statin intake is between 150 mg and 300 mg. 24. The composition of claim 18, wherein the daily intake of tocotrienol is between 250 mg and 500 mg, the daily intake of ionone is between 200 mg and 400 mg and the daily intake of statin is included in 150 mg and 300 mg.