WO2005112967A2 - Anticancer activity of chios mastic gum - Google Patents

Abstract

An anti-cancer compound is purified from Chios mastic gum resin by suspending mastic gum resin in a non-acidic, aliphatic hydrocarbon, an aqueous solution containing at least 25 percent water-soluble, non-acidic, aliphatic hydrocarbon, or a mixture or combination thereof, and removing the insoluble fraction. A method for treating cancer cells comprises administering a pharmaceutically effective amount of a fraction of mastic gum resin that inhibits growth of cancer cells. The anti-cancer compound is effective against various types of cancer cells, including human colon cancer cells.

Description

ANTICANCER ACTIVITY OF CHIOS MASTIC GUM

RELATED APPLICATIONS U.S. Provisional Application Serial No.60/572,703, which was filed on May 19, 2004, is incorporated herein by reference.

BACKGROUND OF THE INVENTION The genus Pistacia of the Anacardiaceae plant family has eleven recognized species, many of which yield resin in some degree. However, only two of them, namely Pistacia atlantica and Pistacia lentiscus L. var. Chia, are major sources of resin that can be collected. Both species are cultivated on Chios island, Greece, and yield the resins known as Chian turpentine and mastic gum, respectively. The Pistacia lentiscus L. is a small evergreen shrub native to the Mediterranean countries. However, the variety Pistacia lentiscus L.. var. Chia grows particularly and almost exclusively in the south region of Chios island. Mastic gum is obtained by shallow incisions of the bark or the trunk and main branches of Pistacia lentiscus L. var. Chia with special tools. Mastic gum has been used for a variety of gastric ailments in Mediterranean and Mideast countries for at least 3,000 years. In ancient times, mastic gum was highly revered for its medicinal properties in the relief of dyspepsia and other disorders. The benefits of this naturally occurring resin are now being rediscovered for their antimicrobial effects. The chemical composition of the resins extracted from the insect galls on the plant of almost all species of Pistacia L. has been analyzed, but only a few constituents have been isolated and identified from the acidic and neutral fractions of gum mastic. Several studies have already been published on mastic gum with regard to its positive effects on the gastrointestinal environment, thus gaining respect among the scientific and medical community. Perhaps the most exciting breakthrough to date is that of a recent study showing mastic gum's effectiveness against at least seven different strains of Helicobacter pylori. In several studies using mastic gum on patients with ulcers, the original site of the ulcer was completely replaced by healthy epithelial cells. These results, although not entirely conclusive, do indicate a strong potential role for the Chios mastic gum (CMG) in maintaining a healthy gastrointestinal system, as well as strong rationale for further studies. Although significant progress has been made in the treatment of various types of cancer, this progress is not universal. Some types of cancer remain resistant to treatment, at least in some patients. Even for those cancers which may be treated with a relatively high likelihood of success, there are constantly some individuals for which the usual treatments are unsuccessful. Furthermore, many forms of cancer treatment also have undesirable side effects. A need exists, therefore, for alternate methods and compositions for the treatment of cancer.

SUMMARY OF THE INVENTION The present invention provides in one aspect a product that is obtainable by a method comprising fractionation of mastic gum resin and that has anti-proliferative properties on growing cancer cells. The product may have the characteristics of a substance comprising or comprised in a fraction of mastic gum resin that has anti-proliferative properties on growing cancer cells, wherein the fraction is soluble in a liquid selected from the group consisting of non-acidic, aliphatic hydrocarbons, aqueous solutions containing at least 25 percent water-soluble, non-acidic, aliphatic hydrocarbons, and mixtures and combinations thereof, and wherein the fraction has been separated from the fraction of mastic gum resin that is insoluble in the liquid. The present invention also provides a method for treating cancer cells comprising administering a product having the characteristics of a product obtained by a method comprising fractionation of mastic gum and having an anti-proliferative property. The product may have the characteristics of a substance comprising or comprised in a fraction of mastic gum resin that is soluble in a liquid selected from the group consisting of non-acidic, aliphatic hydrocarbons, aqueous solutions containing at least 25 percent water-soluble, non-acidic, aliphatic hydrocarbons, and mixtures and combinations thereof, and wherein the liquid soluble fraction of mastic gum resin has been separated from the fraction of mastic gum resin that is insoluble in the liquid. Included also in the invention is the use for the manufacture of a medicament for treating growing cancer cells of a product having an anti- proliferative activity and selected from mastic gum resin and products obtainable by a method comprising fractionation thereof. The present invention also provides a method of making a composition. The method comprises suspending mastic gum resin in a liquid to provide a liquid/mastic gum resin suspension and removing that portion of the liquid/mastic gum resin suspension that is insoluble in the liquid to provide an extracted mastic gum resin solution, wherein the liquid is selected from the group consisting of non- acidic, aliphatic hydrocarbons, aqueous solutions containing at least 25 percent water-soluble, non- acidic, aliphatic hydrocarbons, and mixtures and combinations thereof. Other aspects and embodiments of the invention are set forth below and in the claims.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a photograph of three separate six- well plates seeded with human colon cells (HCT116), breast carcinoma cells (MDA-231 ), or prostate carcinoma cells (DU- 145) and grown in the presence of varying concentrations of an ethanol extract of CMG, which was added to the cultures after they were established. Fig. 2 is a photograph of three separate six- well plates seeded with HCT1 16 cells and grown in the presence of ethanol extracts of CMG following establishment of the cell cultures. Fig. 3 is a photograph of stained HCT116 cells that were suspended in Et-CMG containing medium or a control medium, seeded in Petri-dishes, and then grown in the presence or absence of an ethanol extract of CMG. Figs. 4 is a series of photomicrographs of HCT1 16 cells grown in the presence or absence of an ethanol extract of CMG for 48 hours (Frames A-D) or 72 hours (Frames E-H). Fig. 5 is a series of histograms derived from flow cytometry analysis of a pool of attached and non-attached HCT1 16 cells exposed for 96 hours to low (histogram B) and high (histogram C) concentrations of ethanol extracts of CMG. Control cell cultures received no additive (histogram A) or ethanol without CMG (histogram D). Fig. 6 is a photograph of a Western blot of whole cell protein extracts from HCT1 16 cells that were untreated (lane A), treated with increasing concentrations of CMG ethanol extracts (lanes B, C, and D) for 96 hours. Specific antibodies to pro-caspase-3, PARP and β-actin were used for identification of proteins of interest. Fig. 6A is a photograph of a Western blot of whole protein extracts from HCT1 16 cells that were treated with a selected concentration of CMG ethanol extract for various periods of time (lanes F-L). Specific antibodies to pro-caspase-3 and PARP were used for identification of proteins of interest. Fig. 6B is a photograph of a Western blot of whole protein extracts from HCT1 16 cells, HCT1 16/FADD.DN and HCT1 16/Bcl-2 cells, which were untreated (lane A) and treated with a selected concentration of CMG ethanol extracts for increasing periods of time (lanes B-I). Specific antibodies to pro-caspases-8 and -9 and pi 8 were used for identification or proteins of interest and compared to a control. Fig. 7 is a series of electron micrographs of HCT116 cells before and after Et-CMG treatment at low (Frames A and C) and high (Frames B and D) magnification. Fig. 8 is a series of histograms derived from flow cytometry analysis of HCT1 16 cells exposed to CMG extract for between 24 and 144 hours, and control cells which received no CMG treatment. Fig. 9 is a series of histograms derived from flow cytometry analysis of HCT1 16 cells exposed to an ethanol extract of CMG and then released from CMG extract treatment. The cells were analyzed immediately after treatment for 72 hours with CMG (histogram A) or after release from CMG- treatment for 2, 4, 6, 8, 12, 15, 19 and 24 days (histograms B-I, respectively). Fig. 10 is a series of histograms derived from flow cytometry analysis of HCT1 16 cells exposed to 50 μg/ml and 100 μg/ml He-CMG extract for 24, 48, 72 and 96 hours. Control samples received no He-CMG extract. Fig. 1 1 is a series of histograms derived from flow cytometry analysis of HCT1 16 cells exposed to 25 to 200 μg/ml of He-CMG extract for 24 hours. Control samples received no He-CMG extract. Fig. 12 is a series of histograms derived from flow cytometry analysis of HCT1 16 cells (A-F), HCT1 16/p53 cells (G-L), HCT1 16/Bcl-2 cells (M-R) and HCT116/FADD.DN cells (S-X). The cells were untreated, treated for progressive periods of time with a select concentration of ethanol extracts of CMG, and compared with cells in confluent cultures. Fig. 13 is a photograph of a Western blot of whole cell protein extracts from HCT1 16 cells that were untreated (lane A) and treated with a select concentration of CMG ethanol extract for progressive periods of time (B-E), and compared with untreated confluent cells (lane F) and a control (lane G). Expression of p53 and p21 were used to identify proteins of interest. Fig. 14 is a series of histograms derived from flow cytometry analysis of HCT1 16 cells exposed to various concentrations of He-CMG and HEX over the same time periods. Fig. 15 is a graph showing tumor mean weights measured over a period of days in three groups of 5 NOD/SKID mice injected with equal numbers of human colon cancer HCT1 16 cells, where the first group received no treatment, the second group was treated with a HEX suspension for 4 days followed by 3 days without treatment, and the third group was treated with the same HEX suspension for 5 days followed by 2 days without treatment. Fig 16 is a photograph showing the tumor size at day 31 in a mouse from the group of 5 NOD/SKID mice that received no treatment and in a mouse from the group of 5 NOD/SKID mice treated with a HEX suspension for four days followed by 3 days without treatment. The location of the tumors is indicated by arrows. Fig 17 is a graph showing mean body weights measured over a period of days in three groups of 5 NOD/SKID mice injected with equal numbers ofhuman colon cancer HCT116 cells, where the first group received no treatment, the second group was treated with a HEX suspension for four days followed by 3 days without treatment, and the third group was treated with the same HEX suspension for 5 days followed by 2 days without treatment.

DETAILED DESCRIPTION OF THE INVENTION We have found that Chios mastic gum (CMG) can inhibit proliferation ofhuman cancer cells, or induce death of cancer cells by apoptosis. There is therefore disclosed the use for the manufacture of a medicament for treating growing cancer cells of a product which is CMG or has the characteristics of a substance (e.g. a compound or a mixture of compounds) comprised in CMG. Products which have the characteristics of a substance comprising or comprised in CMG may be obtainable by a method comprising fractionation of CMG. The active anti-cancer component of CMG may be at least partially purified or isolated by suspending the mastic gum in a non-acidic, aliphatic hydrocarbon, an aqueous solution containing at least 25 percent water-soluble, non-acidic, aliphatic hydrocarbon, or mixtures or combinations thereof, and removing that portion of the suspension that is insoluble. Examples of suitable solvents for suspension of the mastic gum resin include C i -Cβ alkanes, halogenated C i -Cβ alkanes, sulfinated C l - C6 alkanes, C]-C6 alcohols, aqueous solutions containing at least 25 percent water-soluble Cj -Cβ alcohols, and mixtures and combinations thereof. Specific examples of suitable solvents include methanol, ethanol, dimethylsulfoxide (sulfinylbismethane), hexane, chloroform, and aqueous solutions containing 25 percent ethanol or more. Solutions of 50 percent ethanol are particularly useful, as is 100 percent ethanol. Hexane is also a particularly useful solvent. The product of the process described in the previous paragraph may additionally be subject to one or more additional processing steps and a pharmaceutical formulation may be formed. The formulation may contain one or more pharmaceutically acceptable diluents, excipients and/or carriers as well as the active agent. The CMG is optimally a resin from a plant of the species Pistacia, especially Pistacia lentiscus. Pistacia lentiscus L. is particularly desirable as a source of CMG resin, especially plants of the variety Pistacia lentiscus L. var Chia. The CMG at least partially purified by a method of this invention may be used to treat cancer cells by administering an effective amount of the at least partially purified active component of the resin to the cancer cells. However, the invention is not limited as to the process by which the active anti-proliferative product is obtained: it may be obtained by fractionation/purification of CMG as described herein but it may also be a substance having the characteristics of a product obtained in this way but obtained by another route. The active product may therefore comprise one or more compounds contained in or derived from CMG but provided by a synthetic or semi-synthetic route, or obtained from an alternative natural source. As previously indicated, therefore, the disclosure includes products having anti-cancer properties which are a CMG or comprised in a CMG, irrespective of the actual route of preparation. The products may therefore comprise mixtures of compounds or an individual compound derived from CMG. Such compounds or mixtures may be isolated or separated from CMG by known processes, for example solvent extraction, fractional crystallization, chromatography, e.g. HPLC, and/or other suitable separation processes. Anti-cancer activity may be tested for using conventional in vitro and/or in vivo tests, for example in vitro tests on suitable cell lines or in vivo tests using appropriate animal models. The skilled person will be aware of particular tests appropriate for testing anti-cancer activity. The disclosure therefore includes methods of identifying anti-cancer activity products, comprising subjecting CMG or a fraction thereof to one or more separation and/or fractionation processes and testing the product of such process for anti-cancer activity. The identified product, whether obtained from CMG, another natural source or synthetically, may be used as a medicament. The products may comprise acidic or basic groups. Such products may be administered in the form of pharmaceutically acceptable salts. The pharmaceutically acceptable salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, and in Stahl et al, Eds, "Handbook of Pharmaceutical Salts Properties Selection and Use", Verlag Helvetica Chimica Acta and Wiley-VCH, 2002. The products of the disclosure therefore include such products which are salts. The disclosure includes prodrugs for the active pharmaceutical substances, for example in which one or more functional groups are protected or derivatised but can be converted in vivo to the functional group, as in the case of esters of carboxylic acids convertible in vivo to the free acid (which representation includes tetrahedrol boronate species, as discussed below), or in the case of protected nitrogens. The term "prodrug," as used herein, represents compounds which are rapidly transformed in vivo to the parent compound, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; H Bundgaard, ed, Design of Prodrugs, Elsevier, 1985; and Judkins, et al. Synthetic Communications, 26(23), 4351-4367 (1996). The use of protecting groups is fully described in 'Protective Groups in Organic Chemistry', edited by J W F McOmie, Plenum Press (1973), and 'Protective Groups in Organic Synthesis', 2nd edition, T W Greene & P G M Wutz, Wiley-Interscience (1991). Thus, it will be appreciated by those skilled in the art that, although protected derivatives of the products may not possess pharmacological activity as such, they may be administered, for example parenterally or orally, and thereafter metabolised in the body to form compounds of the disclosure which are pharmacologically active. Such derivatives are therefore examples of "prodrugs". All prodrugs of the products are included within the scope of the disclosure. The products referred to or featured herein (especially those containing heteroatoms and conjugated bonds) may exist in tautomeric forms and all these tautomers are included in the scope of the disclosure. More generally, many species may exist in equilibrium, as for example in the case of organic acids and their counterpart anions; a reference herein to a species accordingly includes reference to all equilibrium foπns thereof. The products of the disclosure may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the disclosure. Geometric isomers may also exist in the compounds of the present disclosure. The present disclosure contemplates the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond and designates such isomers as of the Z or E configuration, wherein the term "Z" represents substituents on the same side of the carbon- carbon double bond and the term "E" represents substituents on opposite sides of the carbon—carbon double bond. The disclosure therefore includes all variant forms of the defined products, for example any tautomer or any pharmaceutically acceptable salt, ester, acid or other variant of the defined compounds and their tautomers as well as substances which, upon administration, are capable of providing directly or indirectly a compound as defined above or providing a species which is capable of existing in equilibrium with such a compound. The products of the invention may be administered orally or parenterally, e.g. intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation. The products may be administered in the form of pharmaceutical preparations comprising prodrug or active principle either as a free compound or, for example, a pharmaceutically acceptable non-toxic organic or inorganic acid or base addition salt, in a pharmaceutically acceptable dosage form. Depending upon the patient to be treated and the route of administration, the compositions may be administered at varying doses. Typically, therefore, the pharmaceutical compounds of the invention may be administered orally or parenterally ("parenterally" as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intraarticular injection and infusion.) to a host to obtain an anti-cancer effect. In the case of larger animals, such as humans, the compounds may be administered alone or as compositions in combination with pharmaceutically acceptable diluents, excipients or carriers. Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active product(s) that is effective to achieve the desired therapeutic response for a particular patient, compositions, and mode of administration. The selected dosage level will depend upon the activity of the particular product, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the product at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. According to a further aspect of the invention there is thus provided a pharmaceutical composition including a product of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. Pharmaceutical compositions of this invention for parenteral injection suitably comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol or phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars or sodium chloride, for example. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents (for example aluminum monostearate and gelatin) which delay absorption. In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are suitably made by forming microencapsule matrices of the drug in biodegradable polymers, for example polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example. Suitably, oral formulations contain a dissolution aid. The dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable. Examples include nonionic surface active agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkylamine oxides; bile acid and salts thereof (e.g., chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and salts thereof, and glycine or taurine conjugate thereof); ionic surface active agents, such as sodium laurylsulfate, fatty acid soaps, alkylsulfonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface active agents, such as betaines and aminocarboxylic acid salts. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, and/or in delayed fashion. Examples of embedding compositions which can be used include polymeric substances and waxes. The active compounds may also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active product(s), the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl fonnamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof. Compounds of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p 33 et seq. Pharmaceutical formulations may comprise at least one further active principle in addition to one or more products of the invention, for example a further anti-cancer agent, or any product to modulate the activity of the formulation or its side effects.In order to demonstrate the practice of the present invention, the following examples are provided. The following examples are illustrative and should not be viewed as limiting the scope of the invention. An ethanol extract of dry CMG resin (referred to as Et-CMG herein) was produced by suspending pulverized dry resin in ethanol. The Et-CMG extract was provided to cell culture at a final concentration of about 500 μg/ml. The final ethanol concentration was about 0.5 percent or less. Various human cell lines derived from diverse tissues were seeded in Petri dishes, treated with Et-CMG and monitored for extent of proliferation (following staining with crystal blue dye) and apoptosis (following microscopy observations) after 72 hours. The results are summarized in Table I. DURC-1 cells are derived from DU-145 cells and are resistant to 1 μM of 9-nitrocamptothecin. The FADD-DN/Jurkat, casp 8-/Jurkat, and Bcl-2:HL-60 sub-lines are derived from Jurkat and HL-60 cells following genetic manipulation. They are used as tools to identify specific steps/pathways involved in the process of apoptosis. Cell lines were obtained from the American Tissue Culture Collection (ATCC; McLean, VA) or were genetically generated. Control cultures received no additive or 0.5 percent ethanol alone.

Table I Human Cells and Responses to Treatment with Chios Mastic Gum

Cells Brief Description Induction of Induction of Cell Type Growth Arrest of Apoptosis

Fibroblasts normal yes no

HCT 1 16 colon cancer yes

SKOV3 ovarian adenocarcinoma yes

A2774 ovarian adenocarcinoma yes

PA-1 ovarian teratocarcinoma yes

SB-3 malignant melanoma yes

DU-145 prostate carcinoma yes

DURC-1 9NC-resistant DU-145 yes

MDA-MB-231 breast carcinoma yes

Jurkat T-cell leukemia yes

FADD-DN/Jurkat sub-line of Jurkat yes no casp8-/Jurkat sub-line of Jurkat yes no

HL-60 Acute myelogenous leukemia l yes

Bcl-2:HL-60 sub-line of HL-60 yes no

Extracts of CMG resin in various ethanol solutions were prepared as follows. Equal amounts (1 g) of pulverized dry CMG resin were placed in polypropylene tubes and brought to 5 ml in de-ionized water (Extract A), 25% ethanol in water (Extract B), 50% ethanol in water (Extract C) or 100%) ethanol (Extract D). The mixtures were continually agitated for 24 hours at room temperature. Upon termination of agitation, Extracts A and D were clear, whereas, Extracts B and C appeared "cloudy". All extracts were centrifuged to separate soluble (supernatant) from insoluble (pellet) material. Subsequently, soluble material was transferred into new polypropylene tubes and stored at room temperature. The insoluble material remaining in the tubes was air-dried and tubes with air-dried materials were weighed to estimate the amounts of insoluble material in each tube (empty tubes were weighed prior to using for CMG extraction). The measurements and estimates of solubilized amounts are summarized in Table 2. Table II Estimate of soluble CMG material Estimated Percent Soluble Concentration :tract Ethanol Amount (grams (mg/ml) A 0 0.0 0 B 25 0.9 36 C 50 2.1 84 D 100 4.8 192

As shown in Fig. 1 , three 6-well dishes were seeded with human colon (HCT116), breast (MDA-231), and prostate (DU-145) carcinoma cells in RPMI 1640 culture medium (Sigma Co., St. Louis, MO). The wells of each dish contained equal number of cells. The cells were allowed to adhere overnight in a humidified 37°C-incubator in an atmosphere of 5 percent CO . Subsequently, the media and non-attached cells were removed, each well was rinsed with 3 ml of fresh media, and then the adhered cells received 3 ml media containing no additive (well A), 25 μg/ml Et-CMG (well B), 75 μg/ml Et-CMG (well C), 150 μg/ml Et-CMG (well D), 500 μg/ml Et-CMG (well E), and 0.3% ethanol alone (well F). The Et-CMG used was a 50 percent ethanol extract of CMG resin. All dishes were placed in the 37°C-incubator for an additional period of 48 hours, and then media and detached cells were removed. The attached cells were fixed in 100% methanol for 10 minutes, stained with crystal violet dye for 10 minutes, briefly rinsed with PBS, air-dried, and photographed in a Fuji imager. At the highest Et-CMG concentration used (about 500 μg/ml), colon cancer cells were more sensitive than breast and prostate cells (compare untreated wells A and treated wells E). However, Et-CMG still exhibited an effect on breast and prostate cancer cells, with MDA-231 cells being more sensitive than DU-145 cells. Because of the higher sensitivity of HCT1 16 cells to Et-CMG, we chose to utilize these cells for further studies. The toxicity/antiproliferative activity of Et-CMG on colon cancer HCT1 16 cells was tested as follows. Three plates (each consisting of 6 wells) were seeded with equal cell populations, and then allowed to incubate overnight at 37°C. The media was removed, the wells were rinsed with fresh media, and then received media containing Extract B (i.e., 25% ethanol extract), Extract C (i.e., 50% ethanol extract) and Extract D (i.e., 100%) ethanol extract). Since the CMG concentrations in extracts B and C were only estimated, we investigated the activity of CMG components contained in equal volumes of extracts added to various final ethanol concentrations, but not exceeding 0.5% ethanol, in the cell cultures. The results are shown in Fig. 2. Partial inhibition of cell proliferation was observed after Extract B was added to a final concentration of 0.5%> ethanol in the culture (see Extract B; compare well E to control wells A and F). A much greater extent of inhibition/antiproliferative activity was apparent in cultures that received Extract C to final ethanol concentrations of 0.2% (well C), 0.3%_> (well D) and 0.5% (well E). Finally, the most dramatic inhibitory/cytotoxic effect was observed in cultures that received Extract D. In the initial experiment, the Extract D volume added to the culture (to obtain 0.5%) ethanol concentration) resulted in extensive visible precipitation of material (not shown), and we chose to use Extract D volumes that resulted in ethanol concentrations of 0.05% (well C), 0.1 % (well D), and 0.2%> (well E). Comparison with the control cultures (wells A and F) indicated that Extract D was effective at low ethanol concentrations in the cultures, whereas the CMG extract in 50%_> ethanol (Extract C) was effective when added to higher ethanol concentrations to the cultures. Although Extract D was the most effective, its use was hindered by the formation of precipitated material in the culture. On the other hand, an extract similar to Extract C has been used previously in studies of controlling Helicobacter pylori. Therefore, we utilized Extract C in all subsequent studies. Monitoring the cultured HCT116 cells for morphological changes under the microscope, we observed that treatment of cells with Et-CMG resulted in loss of adhesion of most cells and their accumulation in suspension. The percent of detached cells increased as a function of time, that is, the longer the exposure to Et-CMG, the higher the number of detached cells in the culture. Loss of adhesion was induced by Et-CMG, since presence of 0.5% ethanol alone in the culture did not appear to result in cell detachment. The detached cells suspended in the culture media formed aggregates that were difficult to disperse by pipeting alone. The dispersion of this aggregates required the presence of a low concentration of heparin, or a brief treatment with a trypsin solution. In one study, exponentially growing HCT116 cells attached to a substrate were detached by trypsinization and harvested in RPMI medium. Equal volumes of the cell suspension, presumably containing an equal number of cells, were centrifuged to pellet the cells. Subsequently, both cell pellets were suspended in equal volumes of fresh medium without (pellet A) and with (pellet B) 500 μg Et- CMG and placed in Petri-dishes in a 37°C-incubator. After 72 hours of incubation, the dishes were rinsed with PBS, treated with fixative (absolute methanol), stained with crystal violet and photographed. Visual comparison of the stained cultures indicated that the presence of Et-CMG prevented the growth of the cultures cells (compare untreated (-CMG) to treated (+CMG) cells in Fig. 3). Further microscopic examination of untreated and Et-CMG treated cells revealed that the presence of low Et-CMG concentrations resulted in moφhological changes in the cells. Photographs of live cells in Phosphate Buffered Saline (PBS), as well as fixed cells stained with Giemsa stain at low (approximately 50X) or high (approximately 250X) magnification, are shown in Fig.4. Frame A shows untreated cells photographed in PBS. Frame B shows Et-CMG treated cells (treated for 48 hours) photographed in PBS. Frame C shows untreated cells after fixing and staining with Giemsa stain. Frame D is a photomicrograph of Et-CMG treated cells (treated for 48 hours) that were fixed and stained with Giemsa stain. Frame E shows untreated cells that were fixed and stained with Giemsa stain and photographed at low magnification. Frame F shows Et-CMG treated cells (treated for 72 hours) that were fixed and stained with Giemsa stain and photographed at low magnification. Frame G shows untreated cells that were fixed and stained with Giemsa stain and photographed at high magnification. Frame H is a photomicrograph of Et-CMG treated cells (treated for 72 hours) that were fixed and stained with Giemsa stain and photographed at high magnification. It is apparent that the cells treated with Et-CMG for 48 hours are less in number, more elongated, and more homogeneous in size than untreated cells, while some of the Et-CMG treated cells have developed hairlike cytoplasmic extensions that contact similar extensions of other Et-CMG treated cells in the culture (Fig. 4, frames A-D). The moφhological differences are more dramatic after 72 hours of Et-CMG treatment (Fig. 4, frames E-H) with nearly all treated cells bearing smaller nuclei with less pronounced nucleoli, while the cytoplasmic extensions and their contacts serve as bridges with the neighboring cells (noted with arrowheads in Fig. 4, frame H). Apoptosis is the biochemical mechanism of death in cells exposed to chemotherapeutic drugs and other agents. In this regard, we have obtained results of studies that demonstrate that Et- CMG induces apoptosis in human colon cancer HCT116 cells in vitro. Cell cycle perturbations and induction of apoptosis have been assessed by the methodology of flow cytometry. Histograms derived from flow cytometry analysis of a pool of attached and non-attached HCT1 16 cells exposed to Et-CMG are shown in Fig. 5. HCT1 16 cells were exposed for 96 hours to 200 μg Et-CMG/ml (Fig. 5, histogram B) and 500 μg Et-CMG/ml (Fig. 5, histogram C) using Extract C as the source of Et-CMG. Control cultures received no additive (Fig. 5, histogram A) or 0.5%> ethanol alone (Fig. 5, histogram D). The designations in the histograms of Figs. 5 and 8-1 1 are: G1 =G1+G0 cells; G2= G2+M cells; and AP= apoptotic cells. It is apparent that the size of the apoptotic (AP) fraction was dependent on the CMG concentration in the culture rather than the percent of ethanol (see histograms B and C), since ethanol alone did not induce apoptosis (histogram D), and the cell cycle pattern was very similar to that of untreated cells (histogram A). This indicates that Et-CMG exerts its apoptotic ability by introducing, directly or indirectly, DNA damage in the cancer cells. In addition to utilizing flow cytometry, apoptosis was biochemically investigated by detecting the proteolytic degradation (i.e., activation) of pro-caspase-3 in Et-CMG treated HCT1 16 cells. Caspase-3 activation can be detected in all forms of apoptotic pathways, that is, upon induction either of the mitochondrion-dependent or the death receptor-dependent pathway. Proteolytic processing of pro-caspase-3 to yield activated caspase-3 is a late event of the apoptosis mechanism, and is followed by caspase-3 -targeted and cleaved vital cellular acromolecules including the enzyme poly(ADP- ribose) polymerase, PARP. In this regard, we investigated the cleavage of PARP in untreated and Et- CMG treated HCT1 16 cells. The 116 kD-intact and 85 kD-cleaved PARP were analyzed by Western blotting using an antibody that recognizes these two macromolecules. The results are shown in Fig. 6. Processing of pro-caspase-3 was observed after treatment with 100 μg/ml (lane B), 200 μg/ml (lane C) and 500 μg/ml CMG (lane D), but not in untreated (lane A) and ethanol-treated (lane E) cells after 96 hours (Fig. 6). Specifically, 100 μg/ml CMG resulted in detectable pro-caspase-3 reduction as a consequence of proteolytic processing. Pro-caspase-3 reduction was more extensive after treatment with 200 μg CMG/ml, and was dramatically pronounced after treatment with 500 μg CMG/ml, but was not observed in extracts from untreated and ethanol-treated control cells. The results shown in Fig. 6 were derived upon analysis of equal amounts of total cell protein extracts as assessed by the presence of similar levels of the control protein, β-actin, in untreated, Et-CMG treated and alcohol treated cells. The specificity of the proteolytic degradation of PARP was assessed by the demonstration that the control protein, β-actin, was intact at similar levels. Fig. 6A similarly depicts the onset of pro-caspase-3 degradation (i.e., activation) and PARP degradation in HCT1 16 cells treated with CMG/0.5%> ethanol for various periods of time (lanes F-L). Substantially identical amounts of pro-caspase-3 were present and exponentially grown untreated and 24 hours treated cells (lanes F, G), that is, cells attached to the substrate. But a dramatic pro- caspase-3 reduction, as a result of specific degradation, was detected at 72 hours, 120 hours, and 144 hours of treatment in cells that were detached from the substrate (lanes H-J). No pro-caspase-3 degradation was apparent in untreated confluent, and camptothecin (CPT)-arrested senescent cells (lanes K-L) that were also attached to the substrate. Consistent with these observations were also findings that only intact PARP was present in exponentially grown untreated cells (lane F) but a small amount of the 85-kD cleaved PARP product was present in 24 hour treated cells (lane G). Extensive processing of intact PARP was apparent at 72, 120, and 144 hours of CMG-treatment (lanes H-J). Processing of PARP increased as a function of treatment, and resulted in complete cleavage of intact 1 15-kD PARP to the 85-kD cleaved product. In contrast, cleaved 85-kD PARP was absent in untreated confluent Gl -arrested cells, apparently because of spontaneous death of senescent HCTl 16 cells after 144 hours of CPT-treatment (lane L). Fig. 6B depicts processing of caspases-8 and -9 on CMG treated cells. There, flow cytometry results indicated that Bcl-2 over-expression or an impairment in the death receptor-dependent apoptotic pathway does not protect HCTl 16 cells from CMG-induced apoptosis (Fig. 12). On the other hand, CMG induced activation of caspase-3 (Fig. 6a), a late enzymatic event in the mitochondrion- and/or death receptor-dependent pathways. Therefore, we investigated the processing (i.e., activation) of caspases-8 and -9 that have been associated with the pathways dependent on death receptors and mitochondria, respectively. Processing of pro-caspases-8 and -9 were compared in CMG-treated HCTl 16, HCTl 16/FADD.DN and HCTl 16/Bcl-2 cells (Fig. 6b). Only the intact pro-caspase-8 doublet was present in untreated HCTl 16 cells (lane A), whereas a small amount of pi 8, a product of pro-caspase-8, was detected at 24 hr of treatment (lane B), but the pi 8 amount dramatically increased at 72 hr of treatment (lane C). Increases in pi 8 were concomitant with decreases in pro-caspase-8. A similar processing pattern of pro-caspase-8 was also detected in HCTl 16/Bcl-2 cells treated with CMG for 24 hr and 72 hr (lanes D,E,F). Further, a much less extensive processing of pro-caspase-8 was detected in CMG-treated HCTl 16/FADD.DN cells (lanes G,H,I), but apparently this insufficient processing was unable to protect the cells from CMG-induced death observed by flow cytometry (Fig. 12). In a parallel experiment and using the same cell protein extracts, we detected similar patterns of pro-caspase-9 processing, observed as decreases in pro-caspase-9, in CMG-treated HCTl 16 and HCTl 16/Bcl-2 cells, and a less extensive processing in CMG-treated HCTl 16/FADD.DN cells (Fig. 6a). However, inadequate processing (i.e., insufficient activation) of pro-caspase-9 in HCTl 16/FADD.DN cells does not appear to confer significant resistance to CMG-induced death as assessed by flow cytometry (Fig. 12). It should be noted that the specific processing of pro-caspases-8 and -9 was confirmed by the absence of quantitative changes in the amounts of other cellular proteins that also served as control for equal protein analysis (Fig. 6b). Fig. 7, which is a composite of electron micrographs of HCTl 16 cells before and after Et-CMG treatment, shows structural changes in major cellular organelles after treatment for 72 hours with 500 μg Et-CMG extract (scale bar = 1 μm). Compared to untreated control cells (Frames A and B), Et-CMG treated cells (Frames C and D) exhibit loss of the smooth continuity of the cell membrane (cm), appearance of lobulated nuclei (nu), heterochromatin (ch) localization along the inner surface of the nuclear envelop (ne), increased and pronounced nucleolar material (nm), disintegration or loss of organization of the mitochondrial (mi) structure, and appearance of vacuoles (v) of various sizes as well as secretory vesicles (sv). These moφhological changes characterize dying cells. Induction of apoptosis by Et-CMG is time-dependent. HCTl 16 cells were seeded in Petri-dishes and were exposed to a standard (500 μg) Et-CMG concentration for various periods of time. Control cells received no Et-CMG. Untreated and Et-CMG treated cells were subjected to flow cytometry analysis for detection of apoptosis. The histograms in Fig. 8 demonstrate the detection of a small apoptotic (AP) fraction after 72 hours of treatment (histogram D), and subsequent increase of this fraction as treatment was extended for 96 hours (histogram E), 120 hours (histogram F) and 144 hours (histogram G). Untreated cells remained in culture for 96 hours (histogram A). It should be noted that the cells remained attached to the substrate after 24 hours (histogram B) and 48 hours (histogram C) of Et-CMG treatment, whereas only detached cells were present in the culture after 72 to 144 hours of treatment. Et-CMG treatment has a long-term effect on HCTl 16 cells, even after the Et-CMG is removed. HCTl 16 cells were treated for 72 hours with 500 μg Et-CMG (Extract C), then detached cells were collected, rinsed in fresh cell culture media without Et-CMG, divided to equal volumes and placed in dishes in a 37°C incubator. Samples of attached /detached cells were harvested after various periods of time of release from Et-CMG exposure and subjected to flow cytometry analysis for detection of cell cycle perturbations (Fig. 9, histograms B-I). Cells treated for 72 hours with Et-CMG, but not subsequently released in Et-CMG-free culture medium served as control (Fig. 9, histogram A). Under microscopy, the cells were only or mostly detached (histograms A-F), and mostly or only attached (histograms G-I). The results indicate that the 72-hour treated (histogram A) and 2-day released (histogram B) cells were not proliferating, whereas an apoptotic fraction (i.e., dying cells) became apparent after 4 days of CMG-release (histogram C). The apoptotic fraction (AP) continued to increase in the cultures after 6, 8 and 12 days of release from CMG (histograms D-F). The majority of the cells appeared disintegrated under microscopy, whereas a small number of cells appeared to re- attach to the substrate and resume proliferation (histograms G and H) until the culture became confluent and proliferation was ceased. These results demonstrate that upon treatment with CMG, the majority of HCTl 16 cells die by apoptosis even after removal of CMG from the culture, but a small number of non- dying cells eventually resumes proliferation. A hexane extract of CMG (He-CMG) also shows apoptotic activity. Pulverized CMG was extracted in hexane, the hexane and the material dissolved in it were removed from the insoluble material and the hexane was evaporated. The extract remaining after evaporation of the hexane solvent is a clear, highly viscous material that is completely soluble in dimethylsulfoxide (DMSO). This material has been termed He-CMG. He-CMG induces apoptosis in HCTl 16 cells comparable to the extent of apoptosis observed after using higher Et-CMG concentrations (Extract C). Established HCTl 16 cells were exposed to He-CMG, harvested, and subjected to flow cytometry analysis as described above for Et-CMG exposed cells. The apoptotic effect of He-CMG is time-dependent, as demonstrated by the histograms shown in Fig. 10. Longer exposure of HCTl 16 cells to He-CMG results in more extensive apoptosis. The study was conducted with two He-CMG concentrations, 50 μg/ml and 100 μg/ml, for 24, 48, 72 and 96 hours. The results demonstrated that both He-CMG concentrations had a similar manner of inducing apoptosis in the cells, that is, both concentrations were more effective after prolonged exposure of the cells. The apoptotic effect of He-CMG on HCTl 16 cells is directly dependent on He-CMG concentration. Cultures containing similar number of cells were treated for a standard period of 24 hours with 25-200 μg/ml He-CMG, and then subjected to flow cytometry analysis for detection of apoptotic fractions. The resulting histograms are shown in Fig. 1 1. The results demonstrate that even 50 μg He-CMG/ml can induce apoptosis, with higher He-CMG concentrations of 100 μg/ml and 200 μg/ml generating more extensive apoptotic fractions. Carrier (DMSO) alone had no effect on the cell cycle of HCTl 16 cells. Previous studies have demonstrated that p53 or p53 function can dramatically attenuate radiation and drug-induced apoptosis in various cell systems. Therefore, we investigated whether p53 is involved in CMG-induced apoptosis in HCTl 16 cells. For this investigation, parental HCTl 16 (p53 expressing) and HCTl 16/p53-/- (not expressing p53) cells were exposed to the same CMG-concentration, and then compared for induction of apoptosis at various periods of treatment using flow cytometry. Samples were harvested every 24 hours for analysis, but only results (histograms) with extensive changes are shown in Fig. 12. As controls, we utilized untreated cells growing exponentially, and untreated cells that were allowed to reach confluence, i.e., Gl -arrest. In Fig. 12, histograms A through F were derived from HCTl 16 cells, and histograms G through L from HCTl 16/p53-/- cells. Treatment of both cell types with CMG for 24 hours resulted in a decrease in the number of cells in S-phase, a concurrent decrease in the G2-fraction, and appearance of readily detected apoptotic (AP) fractions (B,H). It should be reminded that 96% or more of both cell types were attached to the culture substrate at 24 hours of CMG-treatment. At 72 hours, 100% of HCTl 16 and HCTl 16/p53-/- cells were detached from the substrate and contained a large number of cells slowly progressing through or arrested at the S-phase (C,I). Prolonged CMG-treatment for 120 and 144 hours resulted in decreased S- and G2- fractions (D,J) in both cell types, while the cell sub-population arrested in early-S (e-S)-phase was larger in HCTl 16 than HCTl 16/p53-/- cells (compare D with J, and E with K). It is also apparent that the AP-fraction increased as a function of time, with HCT1 16/p53-/- cells including larger AP-fraction than HCTl 16 cells at the same time-point of treatment. The relative extent of the e-S cells is further accentuated upon comparing with histograms of Gl -arrested cells in confluent cultures, that concurrently exhibit lack of S-phase cells, and negligible G2-fractions (F,L). In conclusion, the presence of p53 in HCTl 16 cells confers no resistance to CMG-treatment. This conclusion was further confirmed by investigating the expression of p53 and p21 in control and CMG-Extract Ill-treated HCTl 16 cells (Fig. 13). p53 and p21 are expressed in untreated cells (lane A), but their expression is down-regulated after 24 hour-treatment with CMG (lane B), that is, at the time the cells are accumulating at Gl (Fig. 12, histogram B). Down-regulation of p53 and p21 are also observed in untreated confluent, also G 1 -arrested, cells (lane F), indicating that down-regulation of p53 and p21 is associated with arrest of the cells at G 1. Further, practically no p53 and p21 expression was detected in cells exposed to CMG for 72, 120 and 144 hours (lanes C,D,E, respectively), whereas both p53 and p21 were over-expressed in CPT-treated G2-arrestcd cells (lane G) in agreement with a previous report. Therefore, we further conclude that CMG-induced Gl -arrest is associated with down-regulation or lack of expression of p53 and p21 in HCTl 16 cells. Subsequently, we investigated whether CMG-induced apoptosis is mediated by the mitochondrion-dependent pathway, which is regulated by the quantitative ratio of pro- to anti-apoptotic proteins of the Bcl-2 family. In a recent study, it has been shown that silencing of the anti-apoptotic protein, Bcl-2, induces massive p53-dependent apoptosis in HCTl 16 cells in absence of genotoxic drugs necessary to activate p53. In this context, we hypothesized that over-expression of Bcl-2 in HCTl 16 cells would increase the resistance of these cells to CMG-induced apoptosis. To investigate this possibility, we compared the sensitivity of HCTl 16 and Bcl-2-over-expressing HCTl 16/Bcl-2 cells to CMG-treatment. Histograms derived from cells at various time-points of CMG-treatment indicated that the HCTl 16/Bcl-2 cells were not significantly more resistant than HCTl 16 cells to CMG-induced apoptosis (Fig. 4). For example, the AP-fractions were calculated to be about 12% and 8% in HCTl 16 and HCTl 16/Bcl-2 cells, respectively, at 24 hours of CMG-treatment (B,N), and 17% in HCTl 16 and 12%) in HCTl 16/Bcl-2 cells at 48 hours of treatment (histograms not shown). Finally, no significant differences were observed in the AP-fractions at 120 (not shown) and 144 hours (K,Q) of treatment. These results led to the conclusion that over-expression of Bcl-2 in HCTl 16 cells confers no significant increase in resistance to CMG-induced apoptosis indicating that Bcl-2, that regulates the mitochondrion-dependent mechanism of apoptosis, has no effect on CMG-induced apoptosis. We also investigated whether CMG induces apoptosis via the death receptor-dependent pathway. Execution of this pathway requires that pro-caspase-8 interacts with the adaptor protein, Fas-associated death domain (FADD), recruited and bound to activated death receptors to form a death-inducing signaling complex (DISC). Therefore, absence of FADD will result in absence of caspase-8 activation and lack of execution of the death receptor pathway. For this investigation, we analyzed CMG-induced apoptosis in HCTl 16 and HCTl 16/FADD.DN cells. The latter cells were derived from HCTl 16 cells but do not express functional adaptor protein FADD required for execution of the death receptor-dependent apoptosis mechanism. The histograms of untreated and CMG-treated HCTl 16 (A through F) and HCTl 16/FADD.DN (S through X) cells indicate similar responses of these two cell types to CMG at specific time-points of the treatment (Fig. 12, compare B with T, C with U, D with V, and E with W). Therefore, we have concluded that CMG-induced apoptosis is not mediated by the death receptor mechanism. The He-CMG as described above has a highly viscous sticky portion. We were able to remove this portion by applying a series of sub-fractionations using diethyl ester and methanol filtrations and evaporations in a rotavapour. A final product (HEX) was obtained by centrifugation, with the highly viscous portion (the pelleted material) removable from the yellowish oily supernatant or HEX. The pelleted material is not soluble in DMSO, while the HEX is readily soluble in DMSO. The biological activity of HEX was confirmed by flow cytometry. HEX was dissolved in DMSO. Comparison was made with He-CMG, which was also dissolved in DMSO. HCTl 16 cells were treated with various concentrations of He-CMG and HEX. The cells were subjected to flow cytometry analyses of the cell cycle for observations on possible perturbations and apoptosis. The resulting histograms (see Figure 14) demonstrated a very high similarity in the histograms derived after using similar concentrations of He-CMG and HEX for the same periods of treatment. We finally investigated whether HEX suppressed tumor growth using the human xenografts/mouse model. Equal numbers ofhuman colon cancer HCTl 16 cells were inoculated into 15 NOD/SKID mice. Five million cells were injected in the inside flags of the posterior legs of the animals, and then the animals were randomly separated into three equal groups. Each group was placed in a separate cage. The mice were subsequently observed daily for the appearance of tumors. Treatment with the He-CMG suspension was initiated when the tumors became measurable, that is, 9 days post-inoculuation. One group of tumor-bearing mice received no He-CMG (control group). A second group received treatment for 4 days followed by 3 days without treatment. The third group received treatment for 5 days followed by 2 days without treatment. The He-CMG dose was adjusted to be 200 mg/Kg of body weight in a 0.5 ml volume. Tumor size and body weight measurements were taken and recorded every 3-4 days. Fig. 15 shows suspension of tumor growth in both groups of mice treated with He-CMG, with the 5 day-treatment/2 day-suspension of treatment schedule being more efficient. At day 24, the 4 day-treatment/3 day-suspension of treatment schedule resulted in about 17% inhibition of tumor growth, whereas, the 5 day-treatment/2day-suspension of treatment schedule resulted in 46%_> growth inhibition. As shown in Fig. 17, no significant changes were observed in body weight of the mice indicating that He-CMG was not toxic for 24 days. However, the mice receiving the 5 day-treatment/2 day-suspension of treatment started to die after day 24. Thus, the average tumor growth was suppressed by 34% in the mice receiving the 4 day-treatment/3 day-suspension of treatment schedule with 5 of 5 mice alive, and by 60%> in the mice receiving the 5 day-treatment/2 day-suspension oftreatment schedule with 4 of5 mice alive at day 28, and 1 of 5 mice alive at day 32. Figure 16 shows the tumor size at day 31 in a mouse from the group which received no treatment as compared with the tumor size in a mouse from the group which received the 4 day-treatment/3 day- suspension of treatment schedule. Based upon the foregoing disclosure, it should now be apparent that a purified component of CMG will carry out the objects set forth hereinabove. It is, therefore, to be understood that any variations evident fall within the scope of the claimed invention and thus, the selection of specific component elements can be determined without departing from the spirit of the invention herein disclosed and described.

Claims

1. A product that is obtainable by a method comprising fractionation of mastic gum resin and that has anti-proliferative properties on growing cancer cells, the product having the characteristics of a substance comprising or comprised in a fraction of mastic gum resin that is soluble in a liquid selected from the group consisting of non-acidic aliphatic hydrocarbons, aqueous solutions containing at least 25 percent water-soluble, non-acidic, aliphatic hydrocarbons, and mixtures and combinations thereof, and wherein said fraction has been separated from another fraction of mastic gum resin that is insoluble in said liquid.

2. The product according to claim 1 , wherein said liquid is selected from the group consisting of C 1 -Cβ alkanes, halogenated C] -C6 alkanes, sulfinated C\ -C alkanes, Ci-Cό alcohols, aqueous solutions containing at least 25 percent water-soluble Ci-Cβ alcohols, and mixtures and combinations thereof.

3. The product according to claim 2, wherein said liquid is selected from the group consisting of methanol, ethanol, dimethylsulfoxide, hexane, chloroform, and aqueous solutions containing greater than 25 percent ethanol.

4. The product according to claim 3, wherein said liquid is selected from the group consisting of hexane and aqueous solutions containing at least 50 percent ethanol.

5. The product according to any preceding claim, wherein the mastic gum resin is a product of a plant of the genus Pistacia.

6. The product according to claim 4, wherein the plant is of the species lentiscus.

7. The product according to claim 5, wherein the plant is Pistacia lentiscus L. var. Chia.

8. The product according to any preceding claim, wherein said fraction is soluble in dimethylsulfoxide, and wherein said fraction has been separated from another fraction that is insoluble in dimethylsulfoxide.

9. The product according to any preceding claim which is obtainable by further processing of said fraction.

10. A pharmaceutical formulation comprising a product of any preceding claim.

1 1. The use for the manufacture of a medicament for treating cancer of a product having an anti- cancer activity and selected from mastic gum resin and products obtainable by a method comprising fractionation thereof

12. The use of claim 1 1 wherein the medicament is for treating colon cancer, ovarian adenocarcinoma, malignant melanoma, prostate carcinoma, 9NC-resistant DU-145, T-cell leukemia or acute myelogenous leukemia

13. The use of any of claims 1 1 to 12 wherein the product is obtainable by a method comprising fractionation of mastic gum resin and inhibits growth of cancer cells.

14. The use of claim 13, wherein the product is as defined in any of claims 1 to 9.

15. A pharmaceutical formulation adapted for parenteral administration and comprising a product having an anti-cancer activity and selected from mastic gum resin and products obtainable by a method comprising fractionation thereof for the manufacture of a medicament for treating cancer.

16. A formulation of claim 15 which is adapted for intravenous administration.

17. A method of at least partially purifying a mastic gum resin, the method comprising: suspending mastic gum resin in a liquid selected from the group consisting of non-acidic, aliphatic hydrocarbons, aqueous solutions containing at least 25 percent water soluble, non- acidic, aliphatic hydrocarbons, and mixtures and combinations thereof, to provide a liquid/mastic gum resin suspension; and removing that portion of the liquid/mastic gum resin suspension that is insoluble in the liquid to provide an extracted mastic gum resin solution;

18. The method according to claim 17, wherein said liquid is selected from the group consisting of C1 -C6 alkanes, halogenated C]-C6 alkanes, sulfinated Ci-Cό alkanes, C\-Cβ alcohols, and aqueous solutions of water soluble Ci -C6 alcohols, and mixtures and combinations thereof.

19. The method according to claim 17, wherein said liquid is selected from the group consisting of methanol, ethanol, dimethylsulfoxide, hexane, chloroform, and aqueous solutions containing greater than 25 percent ethanol.

20. The method according to claim 17, wherein said liquid is selected from the group consisting of hexane and aqueous solutions containing at least 50 percent ethanol.

21. The method according to claim 17, wherein said liquid is hexane, and additionally comprising subsequently removing the hexane from the extracted mastic gum resin solution to provide a hexane-extracted mastic gum resin.

22. The method according to claim 21 , additionally comprising washing the hexane-extracted gum resin with diethyl ester and methanol and subsequently removing the diethyl ester and methanol to provide a further purified hexane-extracted mastic gum resin.

23. The method according to claim 21 or claim 22, additionally comprising, after performing any washing of claim 23, resuspending the further purified hexane-extracted mastic gum resin in dimethylsulfoxide.

24. The method according to any of claims 17 to 23, wherein the mastic gum resin is a product of a plant of the genus Pistacia.

25. The method according to claim 24, wherein the plant is of the species lentiscus.

26. The method according to claim 24, wherein the plant is Pistacia lentiscus L. var. Chia.

27. The method according to any of claims 17 to 26 which further comprises additionally processing the product and forming a pharmaceutical formulation.

28. A composition comprising a fraction of a mastic gum resin that has anti-proliferative properties on growing cancer cells, wherein said fraction is soluble in a liquid selected from the group consisting of non-acidic aliphatic hydrocarbons, aqueous solutions containing at least 25 percent water-soluble, non-acidic aliphatic hydrocarbons, and mixtures and combinations thereof, and wherein said fraction has been separated from another fraction of mastic gum resin that is insoluble in said liquid.

29. The composition according to claim 28, wherein said liquid is selected from the group consisting C1 -C6 alkanes, halogenated Cι-C6 alkanes, sulfinated C1 -C6 alkanes, C1 -C6 alcohols, aqueous solutions containing at least 25 percent water-soluble C1 -C6 alcohols, and mixtures and combinations thereof.

30. The composition according to claim 29 wherein said liquid is selected from the group consisting of methanol, ethanol, dimethylsulfoxide, hexane, chloroform, and aqueous solutions containing greater than 25 percent ethanol.

31. The composition according to claim 30, wherein said liquid is selected from the group consisting of hexane and aqueous solutions containing at least 50 percent ethanol.

32. The composition according to claim 28, wherein the mastic gum resin is a product of a plant of the genus Pistacia.

33. The composition according to claim 32, wherein the plant is of the species lentiscus.

34. The composition according to claim33, wherein the plant is Pistacia lentiscus L. var. Chia_.

35. The composition according to claim 28, wherein said fraction is soluble in dimethylsulfoxide, and wherein said fraction has been separated from another fraction that is insoluble in dimethyl sulfoxide.

36. A method for treating cancer cells comprising administering a pharmaceutically effective amount of a fraction of mastic gum resin that inhibits the growth of cancer cells.

37. The method according to claim 36, wherein said fraction, of mastic gum resin that inhibits the growth of cancer cells is soluble in a liquid selected from the group consisting of non-acidic aliphatic hydrocarbons, aqueous solutions containing a least 25 percent water soluble, non-acidic, aliphatic hydrocarbons, and mixtures and combinations thereof, and wherein said fraction of mastic gum resin that inhibits the growth of cancer cells has been separated from another fraction that is insoluble in said liquid.

38. The method according to claim 37, wherein said liquid is selected from the group consisting of C1 -C6 alkanes, halogenated Cι-C6 alkanes, sulfinated Cι-C6 alkanes, Ci-Cβ alcohols, aqueous solutions containing at least 25 percent water-soluble Ci -C6 alcohols, and mixtures and combinations thereof