WO2007038291A1

WO2007038291A1 - Black tea polyphenols and uses thereof

Info

Publication number: WO2007038291A1
Application number: PCT/US2006/037015
Authority: WO
Inventors: Yukihiko Hara; Nagini Siddavaram
Original assignee: Mitsui Norin Co., Ltd; Annamalai University
Priority date: 2005-09-22
Filing date: 2006-09-21
Publication date: 2007-04-05
Also published as: EP1942918A4; EP1942918A1; JP2009509960A

Abstract

Contemplated compositions and methods include a combination of a tea polyphenol composition and lactoferrin and are effective to prevent and/or treat oral cancer at efficiencies significantly higher than treatments with either tea polyphenol compositions or lactoferrin alone. Such improved efficiency is also reflected in various clinical and systemic parameters, including modulated activity of phase I/II enzymes, expression of cytokeratins, NF-kB, Bcl-2, PCNA, and changes in cellular proliferation and induction of apoptosis.

Description

BLACKTEAPOLYPHENOLS AND USES THEREOF

This application claims priority to our copending U.S. provisional patent application with the serial number 60/720007, which was filed September 22, 2005. Field of The Invention

The field of the invention is treatment and/or chemoprevention of oral cancer, and especially by combined administration of tea extracts and lactoferrin.

Background of The Invention

Oral cancer is the fifth most common malignancy worldwide with about 500,000 new cases diagnosed annually. Unfortunately, about 75% of those occur in developing countries and no effective treatment has been found that is both widely available as well as affordable in such countries.

For example, Tanaka et al. reported chemopreventive effect of bovine lactoferrin to at least some degree on 4-nitroquinoline 1 -oxide-induced tongue carcinogenesis in male F344 rats and suggested that the chemopreventive action against tongue tumorigenesis may have been mediated through modification of cell proliferation activity and/or the activities of detoxifying enzymes (Jpn J Cancer Res. 2000 Jan;91(l):25-33). In other examples, various tea polyphenols were reported in various publications as having some antineoplastic effect. Hsu et al observed that p2 IWAFl can be induced by green tea polyphenol EGCG in several cancer cell types, and that p21 WAF 1 is involved in EGCG-induced growth arrest of OSC2 cells, which may facilitate caspase 3-mediated apoptosis. Based on these observations, Hsu hypothesized that expression of functional p2 IWAFl may promote phytochemical-mediated growth arrest and apoptosis in oral carcinoma cells (Anticancer Res. 2005 Jan-Feb;25(lA): 63-7). In further experiments, Hsu further discovered that green tea and selected constituents of green tea selectively induced apoptosis to at least some degree only in oral carcinoma cells, while EGCG was able to inhibit the growth and invasion of oral carcinoma cells (Gen Dent. 2002 Mar- Apr;50(2): 140-6).

EGCG was also reported to play a significant role in promoting oxidative stress as shown by Weisburg et al. Here the authors reported that EGCG acted as a prooxidant, with the cancerous cells being more sensitive to oxidative stress than the normal cells (Basic Clin Pharmacol Toxicol. 2004 Oct;95(4): 191-200). Green tea was also demonstrated to reduce the mean tumor burden and the incidence of dysplasia and oral carcinoma to some degree (Nutr Cancer. 1999;35(l):73-9), and Li et al. presented data indicating that a combination of tea and curcumin significantly decreased oral visible tumor incidence, squamous cell carcinoma incidence, tumor volume, as well as the numbers of dysplasic lesions and papillomas (Wei Sheng Yan Jiu. 2002 Oct;31(5):354-7).

Unfortunately, while most of the reported compositions and methods exhibit at least some chemopreventive or therapeutic effect, various difficulties still remain. Among other things, the concentration of the polyphenols needed to sustain effect are relatively high. Moreover, as metabolic conversion of the polyphenols is relatively fast, serum concentrations will drop quickly in animals and human, rendering the efficacy less than desirable.

Thus, while numerous compositions and methods for treatment or prevention of oral cancer are known in the art, all or almost all of them, suffer from one or more disadvantages. Therefore, there is still a need for improved compositions and methods for chemoprevention and/or treatment of neoplastic diseases, and especially oral cancer.

Summary of the Invention

The present invention is directed to compositions and methods for chemoprevention and/or treatment of oral cancer in which a tea polyphenol and lactoferrin are combined to exhibit significant antineoplastic effect, wherein the compounds in such combinations may be administered alone or together.

In one aspect of the inventive subject matter, a nutraceutical or pharmaceutical product includes a combination of a tea polyphenol composition and lactoferrin in an amount effective that reduces oral cancer at an efficiency that is greater than an efficiency achieved by oral administration of the product with either the tea polyphenol composition or the lactoferrin alone. Most preferably, the polyphenol composition comprises a mixture of a plurality of catechins obtained from black tea and/or green tea, and may also include one or more synthetic catechins. In further preferred aspects, the polyphenol composition is present in an amount of between 100 mg and 600 mg per dosage unit, and the lactoferrin is present in an amount of between 200 mg and 1200 mg per dosage unit, and it is especially preferred that the combination is synergistic. Contemplated nutraceutical products especially include solid preparations (e.g., capsules, tables, snack bars, etc.), but where desired, the polyphenol composition and/or the lactoferrin may also be present in a liquid carrier.

Therefore, method of improving treatment or chemoprevention of oral cancer using either oral administration of a tea polyphenol composition or oral administration of lactoferrin will typically include a step of ascertaining that oral administration of the tea polyphenol composition or oral administration of the lactoferrin reduces oral cancer with a first efficiency. In a second step, information is provided that combined oral administration of the tea polyphenol composition and the lactoferrin reduces oral cancer with a second efficiency that is greater than the first efficiency.

In particularly preferred methods, the tea polyphenol composition comprises a mixture of a plurality of catechins obtained from black and/or green tea, and the lactoferrin is bovine lactoferrin. Typically, the weight ratio in the combined oral administration between the tea polyphenol composition and the lactoferrin is between 1 : 1 and 1:3, and most typically a synergistic ratio. It is still further preferred that the oral administration is coadministration in a single dosage unit form.

Consequently, and viewed from another perspective, the inventors contemplate a use of a combination of a tea polyphenol composition and lactoferrin in the production of a nutraceutical or pharmaceutical product for treatment or chemoprevention of oral cancer. In such uses, the tea polyphenol composition and the lactoferrin are present in a weight ratio of between 1:1 and 1:3, and most typically in a synergistic weight ratio. Moreover, it is typically preferred that the tea polyphenol composition comprises a mixture of a plurality of catechins obtained from at least one of black tea and green tea (or at least one synthetic catechin) and that the tea polyphenol composition and the lactoferrin are formulated into a single dosage unit (e.g., in a nutraceutical or pharmaceutical product).

Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention.

Brief Description of the Drawings

Figure 1 includes two tables listing various tumor and histopathological parameters of animals treated with black tea polyphenol compositions. Figure 2 depicts various photomicrographs of tissues (H and E stained) of animals treated with black tea polyphenol compositions.

Figure 3 is a graph showing quantitative analysis of PCNA expression in animals treated with black tea polyphenol compositions.

Figure 4 depicts various photomicrographs (PCNA stained) of tissues of animals of Figure 3.

Figure 5 is a table listing various biochemical parameters of animals treated with black tea polyphenol compositions.

Figure 6 depicts various graphs indicating enzymatic activities in selected organs of animals treated with black tea polyphenol compositions.

Figure 7 depicts various graphs indicating further enzymatic activities in selected organs of animals treated with black tea polyphenol compositions.

Figure 8 depicts various graphs indicating still further enzymatic activities in selected organs of animals treated with black tea polyphenol compositions.

Figure 9 is a table listing various tumor parameters of animals treated with black tea polyphenol compositions.

Figure 10 is a table listing expression of various tumor associated proteins of animals treated with black tea polyphenol compositions.

Figure 11 depicts various photomicrographs of tissues (stained for PCNA, NF-kB, cytokeratin) of animals treated with black tea polyphenol compositions.

Figure 12 depicts various photomicrographs of tissues (stained for Bcl-2, Cyc-c, Caspase 3) of animals treated with black tea polyphenol compositions.

Figure 13 is a graph depicting Caspase 3 activity in animals of Figure 12.

Figure 14 is a table depicting is a table listing various tumor parameters of animals treated with a combination of black tea polyphenol compositions and lactoferrin. Figure 15 depicts photomicrographs of selected tissues (stained for cytokeratins, PCNA, and p53) of animals treated with a combination of black tea polyphenol compositions and lactoferrin.

Figure 16 is a table depicting quantitative results for the animals of Figure 15.

Figure 17 illustrates Western blots and their densitometric analysis of selected proteins of animals treated with a combination of black tea polyphenol compositions and lactoferrin.

Figure 18 is a table listing results for measurement of micronucleated polychromatic erythrocytes in animals treated with a combination of black tea polyphenol compositions and lactoferrin.

Figure 19 is a table depicting is a table listing further tumor parameters of animals treated with a combination of black tea polyphenol compositions and lactoferrin.

Figure 20 depicts various graphs indicating enzymatic activities (Cyt-C, GST, diaphorase) in selected organs of animals treated with a combination of black tea polyphenol compositions and lactoferrin.

Figure 21 depicts various graphs indicating various biochemical parameters (TBARS, LOOH, CD) in selected organs of animals treated with a combination of black tea polyphenol compositions and lactoferrin.

Figure 22 depicts various graphs indicating further biochemical parameters (GSH, GSSG, GSH/GSSG ratio,GPx) in selected organs of animals treated with a combination of black tea polyphenol compositions and lactoferrin.

Figure 23 depicts graphs illustrating viability of human cells (cancer and normal) as a function of exposure to lactoferrin, green tea and black tea polyphenols.

Figure 24 depicts graphs illustrating viability of human cells (cancer and normal) as a function of exposure to combinations of lactoferrin with green tea or black tea polyphenols. Figure 25 depicts photomicrographs of cells stained for apoptotic micronuclei as a function of treatment with combinations of lactoferrin with green tea or black tea polyphenols and a quantitative analysis of same.

Figure 26 depicts various graphs illustrating the cell cycle distribution of human oral cancer cells treated with combinations of lactoferrin with green tea polyphenols.

Figure 27 depicts various graphs illustrating effects of combinations of lactoferrin with green tea polyphenols on ROS and mitochondrial membrane potential in human oral cancer cells.

Figure 28 illustrates Western blots and their densitometric analysis of Bcl-2/Bax and Caspase 3 in humal oral cancer cells as a function of treatment with a combination of green tea polyphenol compositions and lactoferrin.

Detailed Description

The inventors have unexpectedly discovered that chemopreventive and/or therapeutic effects of tea polyphenols can be significantly improved by combination of such compounds and compositions with lactoferrin. Among other remarkable results {infra), the inventors discovered that the improved effect is in many cases synergistic and driven by a multitude of underlying effects, including selective triggering of (mitochondrially mediated) apoptosis in cancer cells, reduction in proliferation of cancer cells, modulation of phase IfLl enzymes, and enhanced antioxidant effect.

Therefore, based on the results presented below and further considerations, it is now contemplated that a nutraceutical or pharmaceutical product will include combination of a tea polyphenol composition and lactoferrin in an amount effective that reduces oral cancer. Most typically, such combinations will be implemented such that the combination has an efficiency that is greater than oral administration of the product with the tea polyphenol composition or the lactoferrin alone. Such increased efficiency is most preferably synergistic. Viewed from a different perspective, it is therefore contemplated that a combination of a tea polyphenol composition and lactoferrin is used in the production of a nutraceutical or pharmaceutical product for treatment or chemoprevention of oral cancer. In further particularly preferred aspects of the inventive subject matter, the inventors contemplate a method of improving treatment or chemoprevention of oral cancer (wherein such method uses either a tea polyphenol composition or lactoferrin alone) in which in one step it is ascertained that oral administration of the tea polyphenol composition or the oral administration of the lactoferrin reduces oral cancer at a first efficiency. In a further step of preferred methods, information (e.g., printed, displayed, audible) is provided that combined oral administration of the tea polyphenol composition and the lactoferrin reduces oral cancer at an efficiency that is greater than the first efficiency.

With respect to the step of ascertaining that oral administration of the tea polyphenol composition or the oral administration of the lactoferrin reduces oral cancer (e.g., incidence, multiplicity, severity, and/or speed of progression to more malignant form) at a first efficiency, it should be appreciated that there are numerous manners of such ascertainment known in the art. However, most typically such step of ascertaining will be realized using experimental determination as described below or performed in a similar manner. For example, controlled trials using animals, or statistical analysis of oral cancer data in human may be used. Alternatively, or additionally, the step of ascertaining may also include reference to published results (on human or animal; e.g., J Pharmacol Sci. 2005 May;98(l): 41-8, or Nutrition. 2006 Sep;22(9):940-6; Toxicol In Vitro. 2005 Mar; 19(2):231-42, or Clin Biochem. 2005 Oct;38(10):879-86) wherein the published data are preferably but not necessarily identical to the ascertained data with respect to chemical composition, dosage, schedule, and/or route.

Similarly, with respect to the step of providing information that the combined oral administration of the tea polyphenol composition and the lactoferrin reduces oral cancer at an efficiency that is greater than the first efficiency, it is contemplated that all of the known manners of providing information are deemed suitable for use herein. For example, such information may be provided in printed, displayed, and/or audible form. For example, where contemplated methods are used to obtain regulatory approval (e.g., for pharmaceutical and/or nutritional use, or to substantiate claim of efficiency), the step of ascertaining may be performed using animal and/or human trials while the step of providing information may be performed in printed (written and graphic) form in the application for approval. On the other hand, where contemplated methods are used for marketing a combination product comprising a tea polyphenol and lactoferrin, the step of ascertaining and the step of informing may be performed by providing marketing material that (together or separately) makes reference to a publication that elaborates on the first efficiency and a publication that elaborates on the efficiency that is greater than the first efficiency.

Contemplated Compounds

With respect to contemplated polyphenol compositions, it is generally preferred that suitable polyphenol compositions are mixtures of catechins that are derived from a plant. For example, particularly suitable plants for isolation of polyphenols include the Chinese tea plant {Camellia sinensis, and especially the leaves), or grape (Vitis vinifera, and especially the seeds). Particularly preferred polyphenol compositions include those commercially available under the name of polyphenon E (green tea polyphenol preparation) and polyphenon B (black tea polyphenol preparation) from Mitsui Norin Japan. However, it should be appreciated that numerous other compositions may be obtained from a variety of plants so long as such plants provide appreciable quantities of polyphenols.

Among other suitable constituents, it is generally preferred that the plant polyphenol composition will include at least 25 wt%, more typically at least 50 wt%, even more typically at least 75 wt%, and most typically at least 85 wt% of one or more (typically between two and seven) of epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3- gallate (ECG), epicatechin (EC), gallocatechin-3-gallate (GCG), Gallocatechin (GC), theaflavins, theaflavinmonogallate-A, theaflavinmonogallate-B, theaflavindigallate, tannin, etc., each of which may be stereochemically/optically pure or present as a mixture of isomers. It is further generally preferred that the plant polyphenol composition has a reduced caffeine content, typically at a concentration of less than 5 wt%, more typically less than 3 wt%, and most typically less than 1 wt%. Depending on the plant, it should be noted that the weight ratio of the particular polyphenols relative to each other may vary considerably. However, especially preferred plant polyphenol compositions include those that are typical from green tea extracts, black tea extracts, and those in which EGCG is the predominant component (typically at a concentration of at least 30 wt%, more typically at least 40 wt%). Black tea extracts may also be characterized as comprising catechins that are polymerized to polymerized catechins. Most typically, polymerized catechins include theaflavins and thearugabins at various weight ratios and average molecular weights. In further contemplated aspects, the plant polyphenol composition may be prepared such that the composition will include two, or even only one polyphenol. In such preparations it may be advantageous to replace the isolated plant polyphenol with one or more synthetic polyphenols, and especially EGCG. Where desirable, contemplated polyphenol compositions may be enriched for galloylated catechins. It should be noted that the catechins (galloylated and non-galloylated) contemplated herein include optical isomers, chiral centers, and/or stereoisomers, and that all of such forms (and mixtures thereof) are contemplated herein. Still further contemplated polyphenols may also include (e.g., synthetic, non-naturally occurring) polyphenols, which have a structure according to Formula 1

Formula 1

in which R₁, R₂, R₃, R₄, R₃', R₄', and R₅ ¹ are independently H, OH, or M; wherein M is OC(O)R, OC(S)R, OC(NH)R, OR, or R; wherein R is optionally substituted alkyl, alkenyl, alkynyl, alkaryl, or aryl; and wherein R₃" is an optional gallic acid ester radical or H.

With respect to suitable lactoferrin compositions, it is typically preferred that the lactoferrin is bovine lactoferrin, which may be isolated from cow milk or be a recombinant product. Thus, it should be noted that the iron load of the lactoferrin may vary considerably, and all known iron loads are considered herein. For example, lactoferrin loads may be between 0-20% iron saturation, between 20-40% iron saturation, between 4-60% iron saturation, or between 60-100% iron saturation. Alternatively, the lactoferrin may also be isolated from human milk or colostrum, or other mammalian milk source. Once more it should be noted that the lactoferrin from such alternative sources may be recombinant.

In still further contemplated aspects, it should be recognized that various other iron and especially ferric ion complexing agents are also suitable. Particularly suitable compounds include proteins with such chelating capability and non-protein molecules. For example, where the compound is a non-protein, desferoxamine may be employed and where the compound is a protein, ferritin or hemosiderin may be used. Moreover, where the protein is relatively large and the iron-binding domain is characterized, suitable iron-chelators may also include iron-binding fragments.

Contemplated Nutraceutical Compositions

It is generally contemplated that all combinations of polyphenols and lactoferrin for the chemoprevention and/or treatment of oral cancers may be included in edible carriers, and especially in those in which both the polyphenol and the lactoferrin are present in the same edible item (i.e., nutraceutical or food product).

For example, suitable food products comprise those that include or are prepared from a plant material (e.g., grains, fruit, vegetable, berries, etc.) and/or animal material (e.g., beef, pork, lamb, poultry, fish, crustacean, milk, milk product, etc.), wherein such materials may be raw or at least partially processed. Therefore, in further contemplated aspects of the inventive subject matter, the food product will be solid or liquid, and provide at least one nutrient (e.g., carbohydrate, protein, lipid, mineral, vitamin, etc.), fiber, and/or water in orally administrable form. Contemplated combinations of polyphenol compositions and lactoferrin may be added to the food product in solid or liquid form, or provided in a manner where the polyphenol composition is in a first edible carrier and the lactoferrin is in a second edible carrier. Most preferably, the food product is a food product for human consumption and formulated as a liquid, liquid nutrient, or formulated in a capsule, tablet, or powder (most preferably catechins and lactoferrin are formulated as a combined single dosage unit).

For example, where the food product is in solid form, contemplated forms especially comprise dietary supplements formulated as capsules, tablets, or other nurraceutically known solid forms (e.g., as powder). However, numerous alternative solid formulations are also deemed suitable and include ready-to-consume products such as snack bars, chewing gums, lozenges, etc, as well as fermented milk products, etc., to which contemplated compounds have been added (or which have been enriched in contemplated compounds). Similarly, where the food product is in liquid form, contemplated food products especially include tea or grape juice, milk products, carbonated beverages, fruit beverages (e.g., native juice, juice from concentrate, or beverage comprising fruit juice), sports drinks, alcoholic beverages, enriched water, etc. In another example, it is contemplated that the degree of processing contemplated food may vary considerably, and all degrees of food processing are deemed suitable for use herein. For example, where a food product is unprocessed {e.g., harvested fruit or vegetable), it is contemplated that the compounds according to the inventive subject matter may be added as a coating, admixture, solution, injection, or otherwise combined with the food product. On the other hand, where the food product is processed to at least some degree {e.g., physical form altered {e.g., rolled oats), or chemical composition changed {e.g., fruit extract or combination of food products), contemplated compounds may be added as a coating, as an admixed ingredient, or may be increased by virtue of the processing.

Depending to the particular type of food product and/or processing, it should therefore be appreciated that contemplated polyphenol compositions and lactoferrin may be added as isolated, individual compounds, and/or as mixtures of individual compounds. Thus, such compounds may be relatively pure, or present as a fraction that is enriched in one or more of such compounds. Alternatively, or additionally, the concentration of contemplated polyphenol compositions and/or lactoferrin in the food products may also be increased by virtue of the processing step {e.g., process that increases concentration of contemplated compounds). It should further be appreciated that admixture of contemplated compounds to food products may be performed in numerous manners, and it is contemplated that all known manners are suitable for use in conjunction with the teachings presented herein.

With respect to the amount of contemplated polyphenol compositions and lactoferrin in the food products, it is generally preferred that the amount of such compounds is such that the combination has an efficiency that is greater than oral administration of the product with the tea polyphenol composition or the lactoferrin alone. Therefore, polyphenol compositions will typically be present in a range of between about 200 mg and 1200 mg, and more typically between 400 mg and 800 mg per daily dosage (which may be administered in one to several units). However, in alternative aspects, lower amounts of polyphenol compositions are also deemed suitable {e.g., between 100 mg and 200 mg, or even less, especially in form of a lozenge or chewing gum). Similarly, higher amounts are also considered, and such amounts will typically be in the range of between about 1200 mg and 2000 mg (and even higher). The term "about" as used herein in conjunction with a numeral refers to a range of that numeral +/- 10%, inclusive, of the numeral.

I l Likewise, lactoferrin quantities may vary considerably and will typically be in the range of about 400 mg and 3000 mg, and more typically between 800 mg and 2000 mg per daily dosage (which may be administered in one to several units). However, in alternative aspects, lower amounts of polyphenol compositions are also deemed suitable (e.g., between 200 mg and 400 mg, or even less, especially in form of a lozenge or chewing gum). Similarly, higher amounts are also considered (e.g., in beverages), and such amounts will typically be in the range of between about 3000 mg and 5000 mg (and even higher). Therefore, it should be appreciated that especially preferred ratios of polyphenols to lactoferrin will be in the range of about 1:1 to about 1:3, and even more preferably about 1:2. Lactoferrin may be administered together with the polyphenol, but administration of lactoferrin in a liquid form and polyphenols in solid form is also considered suitable herein. In most circumstances, it is preferred that the weight ratio of the polyphenol to lactoferrin is a synergistic weight ratio (see experiments below). Therefore, in especially preferred food products, the polyphenol composition is present in an amount of between 100 mg and 600 mg per dosage unit, and the lactoferrin is present in an amount of between 200 mg and 1200 mg per dosage unit. In still further aspects of the inventive subject matter, it should be recognized that contemplated food products may include additional components with at least perceived or demonstrated nutraceutical value. For example, especially preferred additional components will include those known or alleged to reduce oxidative stress, improve immune status, etc.

Contemplated Pharmaceutical Compositions

It is generally contemplated that all combinations of polyphenols and lactoferrin may also be administered as pharmaceutical formulations, wherein the administration of the polyphenol and the lactoferrin may be together in a single dosage unit, or separately. Regardless of the particular formulation, it is preferred that the polyphenols and/or lactoferrin is admixed with a pharmaceutically acceptable carrier. Depending on the particular use, it should be recognized that formulation, route, and/or administration schedule may vary considerably, and it is generally contemplated that the specific formulation, route, and/or administration is not limiting to the inventive subject matter.

Therefore, appropriate formulations include formulations for oral, parenteral, and/or topical (including nasal, buccal, and sublingual) administration, and it is further preferred that contemplated formulations are in unit dosage form. It is still further preferred that the amount of contemplated polyphenols and/or lactoferrin that is combined with a carrier to form a unit dosage form will be the amount that produces the chemopreventive and/or therapeutic effect. Thus, the percentage (%wt) of the active ingredient will typically range from about 10% to about 99% of the total weight, preferably from about 20% to about 95%, more preferably from 50% to about 90%, and most preferably from about 70% to about 90%.

It should be appreciated, however, that the administered dose of the pharmaceutical composition will vary considerably, and a particular dose will at least in part depend on (a) the amount of polyphenols and lactoferrin which are effective to achieve a desired therapeutic response, (b) the formulation of contemplated compounds, (c) the route of administration, and (d) other factors, including age, sex, weight, general health, and prior medical history of the patient being treated. A person of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.

It is generally preferred that the daily dose of contemplated compounds will typically correspond to the amount of the compound which is the lowest dose effective to produce a desired therapeutic effect. Such an effective dose will generally depend upon the factors described above. Therefore, doses of the compounds according to the inventive subject matter will range from about 0.5 mg to about 100 mg per kilogram of body weight per day, more preferably from about 1.0 to about 50 mg per kg per day, and still more preferably from about 2.5 to about 25 mg per kg per day. Thus, a unit dose of polyphenols will typically range from about 100 mg to about 5000 mg, more preferably from about 200 mg to about 1200 mg, and most preferably from about 200 mg to about 800 mg. Similarly, the unit dose of lactoferrin will typically range between 400 mg and 3000 mg, and more typically between 800 mg and 2000 mg per day. Where desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

Exemplary Oral Formulations: It is generally preferred that the pharmaceutical combinations according to the inventive subject matter will be orally administered (individually or separately), and all known forms of oral administration are deemed suitable for use herein, including solid and liquid forms. For example, solid oral forms include capsules, tablets, lozenges, powders, while preferred liquid oral forms include solutions or suspensions in suitable medium (typically aqueous solution). Exemplary suitable pharmaceutically acceptable carriers include fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and/or silicic acid), binders (e.g., alginates, gelatin, carboxymethylcellulose, or polyvinyl pyrolidone), humectants (e.g., glycerol), disintegrating agents (e.g., agar-agar, calcium carbonate, or potato or tapioca starch), solution retarding agents (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium salts), wetting agents (e.g., cetyl alcohol, glycerol monostearate), absorbents (e.g., kaolin, bentonite clay), lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols), coloring agents, buffers, etc. Contemplated oral solid dosage may also be formulated to provide slow or controlled release of the active ingredient (e.g., using hydroxypropylmethyl cellulose in varying proportions to provide a desired release profile, other polymer matrices, liposomes and/or microspheres). It should be appreciated that preparation of contemplated oral solid dosage forms is well known in the art, and all of the known methods are deemed suitable for use in conjunction with the teachings presented herein.

Liquid dosage forms for oral administration of contemplated compounds may be prepared as pharmaceutically acceptable emulsions, micro-emulsions, solutions, suspensions, syrups and elixirs. Therefore, and depending on the particular formulation, the liquid dosage forms may also contain inert diluents, including water or other aqueous and non-aqueous solvents, solubilizing agents and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, etc), suspending agents (e.g., ethoxylated isostearyl alcohols, polyoxyethylene sorbitol), oils (e.g., cottonseed, corn, germ, olive, etc.), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, and further known pharmaceutically acceptable liquid components.

Exemplary Parenteral Formulations: It is generally contemplated that the combinations according to the inventive subject matter may be prepared in a formulation for parenteral use, and especially contemplated parenteral formulations will be liquid formulations for injection. Therefore, appropriate formulations will generally include a pharmaceutically acceptable solvent (e.g., sterile isotonic aqueous or non-aqueous solution), and/or may be prepared as a dispersion, suspension, or emulsion. Alternatively, parenteral formulations may also be provided as a kit that includes contemplated compounds and other components that may be reconstituted to a liquid product prior to use.

Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, 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.

Contemplated Uses

It is generally preferred that the compositions and methods according to the inventive subject matter will be employed in the chemoprevention and/or treatment of oral cancers. Therefore, it should be appreciated that suitable conditions include pre-neoplastic conditions as well as neoplastic conditions which may or may not be metastatic. For example, preneoplastic oral conditions include sores (of viral and non- viral origin), and preneoplastic lesions. Typical oral and/or pharyngeal cancers especially include squamous cell carcinomas, commonly on the floor of the mouth, cheek lining, gingiva, and/or palate.

It should be noted that administration of contemplated compounds and compositions may be purely prophylactic or may commence at the first diagnosis of a preneoplastic lesion or even cancer. Therefore, typical administrations will be preferably long-term. For example, daily administration over at least 30 days, more typically at least 60 days, and most typically at least 180 days are especially preferred. However, in alternative aspects, the administration need not be daily, but may be between once weekly (or even less frequent) to three time weekly or more.

Experiments

(I) Effects of Black Tea Polyphenols Alone

We evaluated the chemopreventive effects of black tea polyphenols (Polyphenon B) administration during the pre-initiation phase of 7,12 dimethylbenz[a]anthracene (DMBA)- induced hamster buccal pouch (HBP) carcinogenesis. The expression of proliferating cell nuclear antigen (PCNA) in the buccal pouch and the concentration of lipid peroxides, protein carbonyl and the antioxidant status in the buccal pouch, liver and erythrocytes were used as biomarkers of chemoprevention.

Chemicals Bovine serum albumin, 2-thiobarbituric acid, 2,4-dmitrophenylhydrazine, GSH,5,5'- dithiobis (2-nitrobenzoic acid) (DTNB8), DMBA and 3,3'-diaminobenzidine were purchased from Sigma Chemical Company (St. Louis, MO, USA). PCNA mouse monoclonal antibody and anti-mouse biotin-labelled secondary antibody were purchased from Dako, Carprinteria, CA, USA. Polyphenon B kindly provided by Mitsui Norin Co., Ltd Tokyo, Japan, is a mixture of epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallocatechin-3-gallate (0.2%), free theaflavins (0.32%), theaflavinmonogallate-A (0.14%), theaflavinmonogallate-B (0.15%), theaflavindigallate (0.21%), tannin (35.6%) and caffeine (4.9%). All other reagents used were of analytical grade.

Animals and Diet

All the experiments were carried out with male Syrian hamsters aged 6-10 weeks weighing between 90-11Og obtained from the Central Animal House, Annamalai University, India. The animals were housed five to a polypropylene cage and provided food and water ad libitum. The animals were maintained in a controlled environment under standard conditions of temperature and humidity with an alternating 12 hours light/dark cycle. The animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the ethical committee, Annamalai University. Experimental diet was prepared every day by mixing Polyphenon B to preweighed standard pellet diet (Mysore Snack Feed, Mysore, India) at a concentration of 0.05 per cent. The dose for Polyphenon B used in the present study corresponds to the daily dietary intake of four cups of tea (30-40mg of tea polyphenols per kilogram body weight by humans). The diet was replenished every day and the food consumption was recorded.

Treatment Schedule

The hamsters were randomized into experimental and control groups and divided into 4 groups of 10 animals each. Group 2 animals received diet containing Polyphenon B, four weeks before carcinogen administration when they were 6 weeks of age and continued until the final exposure to carcinogen. At 10 weeks of age, the hamsters in groups 1 and 2 were painted with a 0.5% solution of DMBA in liquid paraffin on the right buccal pouches using a number 4 brush three times a week for 14 weeks. Each application leaves approximately 0.4 mg DMBA. Hamsters in group 1 received no further treatment. Animals in group 3 were given Polyphenon B alone as in group 2. Group 4 animals received basal diet and served as control. The experiment was terminated at the end of 18 weeks and all animals were sacrificed by cervical dislocation after an overnight fast. Before an animal was killed, the right pouch was grossly inspected to evaluate premalignant lesions or tumor development and photographed. The tumor burden was calculated by multiplying the mean tumor volume (4/3 πr3) (r= ¹A tumor diameter in mm) with the mean number of tumors. The buccal pouch and liver tissues were subdivided and variously processed for distribution to each experiment.

Histopathology

The pouch tissues were fixed in 10% formalin, embedded in paraffin and mounted on polylysine-coated glass slides and stained with haematoxylin and eosin. Basal cell hyperplasia, dysplasia and squamous cell carcinoma (SCC9) were diagnosed. Hyperplasia of buccal pouch epithelium indicated by increased number of basal cells was subjectively graded as mild, moderate and severe based on the thickness of the lining epithelium. Dysplasia was characterized by irregular epithelial stratification, increased number of mitotic figures, increased nuclear to cytoplasmic ratio and loss of polarity of basal cells. Histological grading of dysplasia as mild, moderate and severe was based on involvement of one third, half or the entire epithelium respectively. SCC was diagnosed by the invasion of underlying tissues, nuclear pleomorphism and increased mitoses. A tumor that closely resembles its tissue of origin was graded as well-differentiated SCC.

Immunohistochemistry

The tissue sections were deparaffmised by heat at 60oC for 10 minutes, followed by three washes in xylene. After gradual hydration through graded alcohol, the slides were incubated in citrate buffer (pH 6.0) for two cycles of 5 minutes in a microwave oven for antigen retrieval. The sections were allowed to cool for 20 minutes and then rinsed with Tris- buffered saline (TBSlO). The sections were treated for 15 minutes with 3% H2O2 in distilled water to inhibit endogenous peroxidase activity. Nonspecific antibody binding was reduced by incubating the sections with normal goat serum for 25 minutes. The sections were then incubated with PCNA mouse monoclonal antibody at 4oC overnight. The slides were washed with TBS and then incubated with anti-mouse biotin-labelled secondary antibody followed by streptavidin-biotin-peroxidase for 30 minutes each at room temperature. The immunopre cipitate was visualized by treating with 3,3'-diaminobenzidine and counterstaining with haematoxylin. For negative controls, the primary antibody was replaced with TBS. Positive controls for each antibody were also processed simultaneously. The percentage of positive tumor cells expressing these proteins was graded as 0= <5%, 1= 5-25%, 2= 26-50%, 3= 51- 75% and 4= >75% and the intensity of the immunoreactivity was graded as - = absent, + = weak, ++ = moderate and +++ = strong staining. Tumors were considered positive when more than 10% of the tumor cells were stained.

Preparation Of Tissue Hotnogenate And Erythrocyte Ly sate

Fresh tissues were used for biochemical estimations. The buccal pouch and liver tissues after weighing were homogenized in an all glass homogenizer with Teflon pestle and stored in ice until use. Blood samples were collected in heparinised tubes and the plasma was separated by centrifugation at 1000 g for 15 min at 4°C. After centrifugation, the buffy coat was removed and the packed cells washed three times with physiological saline. The erythrocyte samples (0.5 ml) were lysed with 4.5ml of 0.2M phosphate buffer containing 0.1M NaCl, pH 7.4. The hemolysate was separated by centrifugation at 2500 g for 15 min at 2°C. The biochemical assays were carried out immediately.

Biochemical Assays

Lipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances (TBARSl 1), lipid hydroperoxides (LOOH12) and conjugated dienes (CD13). TBARS were assayed in the pouch and liver tissues by the method of Ohkawa et al. and in erythrocytes by the method described by Buege and Aust. Lipid hydroperoxides were estimated by the method of Jiang et al. and conjugated dienes by the method of Rao & Recknagel. Protein oxidation was measured by the method of Levine et al. based on the reaction of the carbonyl group with 2,4-dmitrophenylhydrazine to form 2,4- dinitrophenylhydrazone.Total SOD and Mn-SOD activities were assayed as described by Oberley et al. based on the half-maximal inhibition of nitroblue tetrazolium (NBT14) reduction. Cu-Zn SOD activity was calculated by deducting the activity of Mn-SOD from total SOD activity. The activity of CAT was assayed by the method of Sinha based on the utilization of hydrogen peroxide by the enzyme. GSH was determined by the method of Anderson by measurement of the yellow colour that develops when DTNB is added to compounds containing sulfhydryl groups. Oxidized glutathione (GSSGl 5) was estimated following oxidation of NADH by glutathione reductase (GR16, EC 1.6.4.2) at 340nm according to the method of Anderson. Seleniumdependent glutathione peroxidase (GPxI 7, EC 1.11.1.9) activity was assayed by following the utilization of hydrogen peroxide according to the method of Rotruck et al. Independent GPx activity was assayed following the method described by Lawrence and Burk using cumene peroxide as substrate. The activity of γ-Glutamyl transpeptidase (GGT 18) was assayed by the method of Fiala et al. using gammaglutamyl p-nitroanilide as substrate. GR activity was estimated by the method of Carlberg and Mannervick, using GSSG as substrate and FAD as cofactor. The protein content was estimated by the method of Lowry et al.with bovine serum albumin as standard.

Statistical Analysis

The data are expressed as mean ± standard deviation. The body weights were analyzed by Student's t test. The χ2 -test combined with Yates' correction was used to analyse tumor incidence. Statistical analysis on the data for tumor burden, immunohistochemical analysis and biochemical assays were analyzed using analysis of variance (ANOVA 19) and the group means were compared by the least significant difference test (LSD20). The results were considered statistically significant if the p value was <0.05.

Results

Gross observations : Table 1 in Figure 1 shows the mean body weight, food consumption, tumor incidence, tumor multiplicity and mean tumor burden in different groups. Hamsters in group 1 showed a tendency to be lower in body weight during the experiment and the mean final body weights were decreased significantly (p<0.01) compared to control (group 4). No significant differences in the body weights were observed in groups 2 to 4. The amount of diet consumed in groups 1 to 4 was not significantly different. At the end of the experimental period, the tumor incidence in group 1 was 100% with a multiplicity of 1.8 per hamster and tumor burden of 346 mm³. Administration of Polyphenon B decreased the tumor incidence to 20 per cent with a multiplicity of 0.3 per hamster. Furthermore, the tumors were significantly smaller (mean tumor burden 17.2 mm³) compared to group 1. No tumors were observed in groups 3 and 4.

Histopathological observations: Table 2 in Figure 1 summarizes the histopathological changes in the buccal pouch of hamsters in experimental and control groups. AU the hamsters in group 1 exhibited severe keratosis, hyperplasia, dysplasia and well differentiated SCC. Dietary administration of Polyphenon B to DMBA painted hamsters (group 2) significantly reduced the incidence of SCC as well as hyperplastic and dysplastic lesions. Of the 10 animals, only two developed SCC while four animals showed mild to moderate dysplasia and the remaining four hamsters displayed only moderate hyperplasia. Mild hyperplastic changes were observed in two of the hamsters in group 3. In control animals, the epithelium was normal, intact and continuous. Representative photomicrographs of histopathological changes in each group are shown in Figure 2 depicting photomicrographs of H and E stained regions of buccal pouch mucosa of control and experimental animals. A. Well differentiated SCC with extensive infiltration into connective tissue of group 1 animals after 14 weeks of DMBA treatment (H and E stain, X 10). B. Photomicrograph of buccal pouch epithelium from a group 2 hamster administered DMBA and Polyphenon B exhibiting moderate dysplasia (H and E stain, X 40). C. Photomicrograph of buccal pouch epithelium from a group 3 hamster administered Polyphenon B alone exhibiting mild hyperplasia (H and E stain, X 40). D. Photomicrograph showing normal buccal pouch histology of control animals (group 4; H and E stain, X 40).

Immunohistochemical findings: Figure 3 shows the effect of Polyphenon B on PCNA expression in the buccal pouch mucosa of control and experimental animals. In DMBA painted hamsters, the mean protein expression of PCNA (81%) was significantly higher than in control animals (group 4). Administration of Polyphenon B (group 2) significantly decreased PCNA expression (52%) compared to group 1. No significant changes in the expression of 9 PCNA were observed in group 3 animals compared to control. Representative photomicrographs of immunostaining are shown in Figure 4. Here, the photomicrographs of immunohistochemical staining of PCNA expression in control and experimental animals (x 10) depict: A. Overexpression of PCNA protein in group 1 animals. B. Decreased expression of PCNA protein in group 2 animals. C. Expression of PCNA protein in group 3 animals. D. PCNA expression in control animals.

Biochemical findings: Table 3 in Figure 5 shows the effect of pretreatment with Polyphenon B on lipid peroxidation and protein oxidation. The extent of lipid peroxidation and the formation of protein carbonyl were significantly lower in the buccal pouch and higher in the liver of DMBA painted hamsters (group 1) compared to control. Dietary administration of Polyphenon B (group 2) significantly modulated DMBA-induced changes in lipid and protein oxidation reflected by a significant increase in the buccal pouch with concomitant decrease in the liver compared to group 1. Administration of Polyphenon B alone significantly reduced the extent of tissue lipid peroxidation and protein oxidation in group 3 animals compared to control.

The activities of the antioxidant enzymes SOD (total SOD, Mn-SOD, Cu-Zn SOD) and CAT in the buccal pouch, liver and erythrocytes of control and experimental animals are shown in Figure 6. In DMBA painted animals (group 1), the activities of SODs and CAT were significantly lower compared to control group. Dietary administration of Polyphenon B significantly increased the activities of the antioxidant enzymes in group 2 animals compared to group 1. Administration of Polyphenon B alone significantly increased the activities of SODs and CAT in group 3 animals compared to control (group 4). The changes in the levels of GSH and the activities of GSH-dependent enzymes in the buccal pouch, liver and erythrocytes are presented in Figures 7 and 8. In DMBA painted animals (group 1), the concentration of GSH, GSSG and GSH/GSSG ratio and the activities of GPx (Se-dependent and independent), GR and GGT were increased significantly in the buccal pouch, whereas in the liver and erythrocytes, these antioxidants were significantly decreased compared to control. Dietary administration of Polyphenon B significantly increased all the antioxidants and detoxification enzymes in the buccal pouch, liver and erythrocytes of group 2 animals compared to group 1. Treatment with Polyphenon B alone significantly enhanced GSH and GSH-dependent enzymes in group 3 animals compared to control.

All the hamsters painted with DMBA alone for 14 weeks developed buccal pouch carcinomas associated with increased expression of PCNA, diminished lipid and protein oxidation and enhanced antioxidant status. In the liver and erythrocytes of tumor-bearing animals, enhanced oxidation of lipids and proteins was accompanied by compromised antioxidant defences. Dietary administration of Polyphenon B effectively suppressed DMBA- induced HBP carcinogenesis as revealed by decreased incidence of tumors and PCNA expression. In addition, Polyphenon B modulated lipid and protein oxidation and enhanced the antioxidant status in the pouch, liver and erythrocytes. We suggest that Polyphenon B exerts its chemopreventive effects by inhibiting cell proliferation in the target tissue and modulating the oxidant-antioxidant status in the target as well as in host tissues.

Therefore, it should be recognized that administration of black tea catechins, and especially Polyphenon B modulated the redox status in the target organ and host tissues and significantly suppressed PCNA expression in the buccal pouch. HCPC-hamster cheek pouch carcinoma cell line Polyphenon B reversed the susceptibility to lipid and protein oxidation whilst simultaneously increasing the antioxidant status in the buccal pouch, whereas in the liver and erythrocytes, the extent of lipid and protein oxidation was reduced with elevation of antioxidants. Thus the differential oxidative events induced in the tumor and host tissues by Polyphenon B reflect its inhibitory role on cell proliferation, as evidenced by downregulation of PCNA expression and inhibition of tumor development. These results demonstrate that dietary administration of Polyphenon B exerts significant protection against HBP carcinogenesis by downregulating PCNA expression, modulating lipid and protein oxidation and enhancing the antioxidant status.

We further evaluated the effect of a black tea polyphenol (Polyphenon B (P-B)) on the expression of cytokeratins, and proliferation and apoptosis associated proteins during 7,12- dimethylbenz[a]anthracene induced hamster buccal pouch (HBP) carcinogenesis. As markers for carcinogenesis, we used development of buccal pouch carcinomas that were associated with increased expression of cytokeratin, PCNA, NF-kB, and Bcl-2, and decreased expression of cytochrome C and caspase 3.

Chemicals

DMBA and 3'-diaminobenzidine were purchased from Sigma Chemical Company, St. Louis, MO, USA. All other reagents used were of analytical grade.

Animals

The experiment was carried out with male Syrian hamsters aged 6-10 weeks weighing between 90-110 g obtained from the Central Animal House, Annamalai University, India. The animals were housed five to a polypropylene cage and provided food and water ad libitum. The animals were maintained in a controlled environment under standard conditions of temperature and humidity with an alternating 12 hours light/dark cycle. The animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the ethical committee, Annamalai University. Experimental diet was prepared everyday by mixing P-B to preweighed standard pellet diet (Mysore Snack Feed, Mysore, India) at a concentration of 0.05 per cent. The dose for P-B used in the present study corresponds to the daily intake of four cups of tea (30-40mg of tea polyphenols per kilogram body weight by humans).13 The diet was replenished everyday and the food consumption was recorded. Treatment Schedule

The hamsters were randomized into experimental and control groups and divided into 4 groups of 10 animals each. Group 2 animals received diet containing P-B four weeks before carcinogen administration when they were 6 weeks of age and continued until the final exposure to carcinogen. At 10 weeks of age, the hamsters in groups 1 and 2 were painted with a 0.5% solution of DMBA in liquid paraffin on the right buccal pouches using a number 4 brush three times a week for 14 weeks. Each application leaves approximately 0.4mg DMBA.2 Hamsters in group 1 received no further treatment. Animals in group 3 were given P-B alone as in group 2. Group 4 animals received basal diet and served as control. The experiment was terminated at the end of 18 weeks and all animals were sacrificed by cervical dislocation after an overnight fast. Before an animal was killed, the right buccal pouch was grossly inspected to evaluate premalignant lesions or tumor development and photographed. The tumor burden was calculated by multiplying the mean tumor volume (4/3 πr³) (r= ¹A tumor diameter in mm) with the mean number of tumors. The buccal pouch tissues were subdivided and variously processed for distribution to each experiment. Tissues were fixed in 10% formalin, embedded in paraffin and mounted on polylysine-coated glass slides. One section from each specimen was stained with haematoxylin and eosin. The remaining sections were used for immunohistochemical staining.

Immunohistochemistry

The tissue sections were deparaffmised by heat at 60°C for 10 minutes, followed by three washes in xylene. After gradual hydration through graded alcohol, the slides were incubated in citrate buffer (pH 6.0) for two cycles of 5 minutes in a microwave oven for antigen retrieval. The sections were allowed to cool for 20 minutes, rinsed with Tris-buffered saline (TBS) and treated with 3% H₂O₂ in distilled water for 15 minutes to inhibit endogenous peroxidase activity. Nonspecific antibody binding was reduced by incubating the sections with normal goat serum for 25 minutes. The sections were then incubated with cytokeratin, PCNA and Bcl-2 (DAKO, Carprinteria, CA, USA) mouse monoclonal antibodies and NF-kB and caspase 3 rabbit polyclonal antibodies and cytochrome C monoclonal antibodies (all Neo Markers, USA) at 4°C overnight. The slides were washed with TBS and then incubated with anti-rabbit and anti-mouse biotin-labelled secondary antibody (both DAKO, Carprinteria, CA, USA) followed by streptavidin-biotin-peroxidase for 30 minutes each at room temperature. The immunoprecipitate was visualized by treating with 3,3'- diaminobenzidine and counterstaining with haematoxylin. For negative controls, the primary antibody was replaced with TBS. Positive controls for each antibody were also processed simultaneously. The labeling indices for PCNA were calculated as the number of cells with positive staining per 100 counted cells in three high power fields. The expression of NF-kB, Bcl-2, cytochrome C and caspase 3 were graded as I= 5-25%, 11= 26-50%, IH= 51-75% and IV= >75%, according to the number of positively stained cells per 100 counted cells. The cytokeratin expression was graded as 0= failure to detect the keratin, I= staining confined either to the basal area or some evidence of suprabasal staining, 11= positive staining throughout the basal and/or suprabasal region.

Colorimetric Estimation Of Caspase 3 Activity

DEVD-specific caspase 3 activity was assayed using CASP-3-C colorimetric kit (Sigma, St. Louis Mo, USA) according to the manufacturer's instructions. Cytosolic extracts were prepared by homogenizing tissues in lysis buffer containing 5OmM HEPES (pH 7.4), 5mM CHAPS and 5mM DTT. The supernatant was collected as an enzyme source. The caspase 3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl- Asp- GIu- VaI- Asp-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in release of the p- nitroaniline (pNA) moiety. The concentration of the pNA released from the substrate is calculated from the absorbance values at 405nm or from a calibration curve prepared with defined pNA solutions.

Statistical Analysis

The tumor incidence and grading of NF-kB, cytokeratin, Bcl-2, cytochrome C and caspase 3 were statistically compared using X2 —test. Statistical analysis on the data for tumor burden was carried out using Student's t test. PCNA labeling index and data for colorimetric assay of caspase 3 were analysed using ANOVA followed by LSD. The results were considered statistically significant if the P value was < 0.05.

Results

Table 4 of Figure 9 shows tumor incidence, mean tumor burden and histopathological changes in control and experimental animals. The tumor incidence in group 1 was 100 per cent with a mean tumor burden of 346 mm³. Histologically, HBP tumors induced by DMBA were invasive squamous cell carcinomas with papillary projections of squamous epithelium into the connective tissues. Two of the 10 animals treated with DMBA and P-B developed SCC, while 4 animals showed mild to moderate dysplasia and remaining 4 hamsters displayed moderate hyperplasia. Mild hyperplastic changes were observed in two of the hamsters in group 3. In control animals, the epithelium was normal, intact and continuous.

Table 5 of Figure 10 shows the effect of P-B on PCNA labeling index and expression of NF-kB, cytokeratin, Bcl-2, cytochrome C and caspase 3 in the buccal pouch of control and experimental animals. In DMBA painted animals (group 1) the expression of PCNA, NF-kB, cytokeratin and Bcl-2 was significantly higher and that of cytochrome C and caspase 3 significantly lower than in control animals (group 4). Administration of P-B (group 2) significantly decreased PCNA, NF-kB, cytokeratin and Bcl-2 expression and significantly increased the expression of cytochrome C and caspase 3 compared to group 1. No significant changes in the expression of PCNA, NF-kB, cytokeratin, Bcl-2, cytochrome C and caspase 3 were observed in group 3 animals. While immunostaining of PCNA showed nuclear localization, NF-kB, cytokeratin, Bcl-2, cytochrome C and caspase 3 were found in the cytoplasmic region. Representative photomicrographs of immunostaining are shown in Figures 11 and 12.

Figure 13 illustrates the activity of DEVD-specifϊc caspase 3 in the buccal pouch in control and experimental hamsters. In DMBA painted animals (group 1), caspase 3 activity was significantly reduced as compared with control (group 4). Treatment with P-B significantly increased enzyme activity in group 2 animals as compared with group 1. In animals administered P-B alone (group 3), the activity of caspase 3 was not significantly different from that in control.

Therefore, it should be noted that dietary administration of black tea polyphenols, and particularly P-B reduced the incidence of DMBA- induced HBP carcinomas and preneoplastic lesions. P-B also significantly downregulated the expression of PCNA, NF-kB and Bcl-2 and upregulated cytochrome C, caspase 3 and cytokeratins in the buccal pouch. The data strongly suggest that P-B acts as a suppressing agent and exerts its antineoplastic property by downregulating PCNA, NF-kB and Bcl-2 and upregulating cytochrome C, caspase 3 and cytokeratin. Interestingly, P-B induced apoptosis in tumor cells but failed to do so in normal cells. Hsu et al. found that EGCG induces growth inhibition of transformed cells by activating the apoptotic pathway without affecting normal growth suggesting that the protective effects on normal cells might be brought about by p57, a molecule closely associated with cell differentiation. These findings support the hypothesis that dietary agents that drive tumor cells to undergo apoptosis but direct normal cells towards a survival pathway are ideal chemopreventive agents.

(II) Effects of Black Tea Polyphenol-Lactoferrin Combinations

We evaluated the antiproliferative and apoptosis inducing effects of bovine lactoferrin (bLF) and black tea polyphenol (Polyphenon-B: P-B) combinations on 7,12-dimethylbenz- [a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. The expression of cytokeratins and other markers, including proliferating cell nuclear antigen (PCNA), NF- kB, mutant p53, Bcl-2, Bax, Fas, and caspase 3 was used as markers for chemoprevention of oral cancer.

Chemicals

DMBA was purchased from Sigma Chemical Company, St. Louis, MO, USA. bLF (lot No. 020119) of purity >96.2% was obtained from Morinaga Milk Industry Co., Ltd, Tokyo, Japan. Black tea polyphenols (Polyphenon-B) were kindly provided by Mitsui Norin Co., Ltd., Tokyo, Japan. The composition of Polyphenon-B is same as described previously. It is a mixture of epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallocatechin-3-gallate (0.2%), free theaflavins (0.32%), theaflavinmonogallate-A (0.14%), theafiavinmonogallate-B (0.15%), theaflavindigallate (0.21%), tannin (35.6%) and caffeine (4.9%). All other reagents used were of analytical grade.

Animals and Diets

The experiment was carried out with male Syrian hamsters aged 8-10 weeks weighing 100-11Og obtained from the Central Animal House, Annamalai University, India. The animals housed five to a polypropylene cage were provided food and water ad libitum and maintained under controlled conditions of temperature and humidity with an alternating light/dark cycle. Animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the ethical committee, Annamalai University. Experimental diets were prepared every day by mixing chemopreventive agents alone and in combination to a preweighed standard pellet diet (Mysore Snack Feed Ltd, Mysore, India). The diet was replenished everyday and the food consumption was recorded. Treatment Schedule

The animals were randomized into experimental and control groups and divided into 8 groups of 20 animals each. In group 1, the right buccal pouches of hamsters were painted three times per week with a 0.5 per cent solution of DMBA in liquid paraffin with a number 4 brush. Each application leaves 0.4 mg. Hamsters in group 1 received no further treatment. In Groups 2-4, the right buccal pouches painted with DMBA, as in group 1, received in addition, basal diet containing 0.2 per cent bLF, 0.05 per cent Polyphenon-B and a diet containing combination of 0.2 per cent bLF and 0.05 per cent Polyphenon-B respectively. Animals in groups 5 through 7 were administered bLF, Polyphenon-B alone and in combination respectively. Group 8 animals served as control. The experiment was terminated at 14 weeks and all animals were killed by cervical dislocation after an overnight fast. Before an animal was killed, the right pouch was grossly inspected to evaluate premalignant lesions or tumor development and photographed. The tumor burden was calculated by multiplying the mean tumor volume (4/3πr3) (r= ¹A tumor diameter in mm) with the mean number of tumors. The buccal pouch tissues were subdivided and variously processed for distribution to each experiment. Tissues were fixed in 10% formalin, embedded in paraffin and mounted on poly Iy sine-coated glass slides. One section from each specimen was stained with haematoxylin and eosin. The remaining sections were used for immunohistochemical staining.

Immunohistochemistry

The tissue sections were deparaffmised by heat at 60oC for 10 minutes, followed by three washes in xylene. After gradual hydration through graded alcohol, the slides were incubated in citrate buffer (pH 6.0) for two cycles of 5 minutes in a microwave oven for antigen retrieval. The sections were allowed to cool for 20 minutes and then rinsed with Tris- buffered saline (TBS), and treated with 3% H2O2 in distilled water for 15 minutes to inhibit endogenous peroxidase activity. Nonspecific antibody binding was reduced by incubating the sections with normal serum for 25 minutes. The sections were then incubated with PCNA, p53 and cytokeratin AE1/AE3 mouse monoclonal antibodies (Dako, Carprinteria, CA, USA) at 4oC overnight. The slides were washed with TBS and then incubated with anti-rabbit and anti-mouse biotin labelled secondary antibody (Dako, Carprinteria, CA, USA) followed by streptavidinbiotin- peroxidase for 30 minutes each at room temperature. The immunoprecipitate was visualized by treating with 3,3'-diaminobenzidine (Sigma) and counterstaining with haematoxylin. For negative controls, the primary antibody was replaced with TBS. Positive controls for each antibody were also processed simultaneously. The labeling index for PCNA was expressed as the number of cells with positive staining per 100 counted cells. The data for cytokeratins was graded as I = staining confined either to the basal area or some evidence of suprabasal staining, II = positive staining throughout the basal and/or suprabasal region. p53 expression was graded as I = 5-25%, II = 26-50%, III = 51- 75% and IV = >75%, according to the number of positively stained cells per 100 counted cells.

SDS-PAGE and Western Blot Analysis

Approximately, 50 mg of each tissue sample was subjected to lysis in a sample buffer containing 62.5 mM Tris (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol and bromophenol blue. The protein concentrations of lysates were determined by Bradford method. SDS-PAGE was performed using equivalent protein extracts (55 μg) from each sample according to Laemmli. The resolved proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Immobilion, Millipore, Bedfore, MA, USA). The membranes were incubated in TBS (150 mM NaCl/50mM Tris, pH7.4) containing 5% nonfat dry milk to block nonspecific binding sites for 1 h. The blots were incubated with 1:1000 dilution of anti Bcl-2, Bax, p53, NF-kB, Fas, and caspase 3 antibody (NeoMarkers, USA) overnight at room temperature. The blots were extensively washed with TBS containing 0.1% Tween-20 (TBS-T) and then incubated with 1:1000 dilution of horseradish peroxidase- conjugated secondary antibodies (Santa Cruz Biotechnology, CA, USA) for 30-45 min at room temperature. After extensive washes in TBS-T, the proteins were visualized by treating with 3,3'-diaminobenzidine (Sigma). Densitometry was performed on IISP flat bed scanner and quantitated with Total Lab 1.11 software.

Colorimetric Estimation of Caspase 3 Activity

Statistical Analysis

The data are expressed as mean ± SD. The χ2 -test combined with Yates' correction was used to analyze the tumor incidence. Statistical analysis on the data for tumor burden, PCNA labeling index and densitometric analysis were analysed using analysis of variance (ANOVA) and the group means were compared by the Tukey-Kramer test. Statistical analysis on the data for CKs and p53 were analysed using χ2 test. The results were considered statistically significant if the p value was <0.05. The nature of interaction between the combined effects of bLF and Polyphenon-B was evaluated as described by Yokoyama et al. In brief, the expected value of combined effect between bLF treatment and Polyphenon-B treatment was calculated as [(observed bLF treatment value)/(control value)] x [(observed Polyphenon-B treatment value)/(control value)]; and the combination index calculated as the ratio of expected value/observed value. A ratio of >1 indicates a synergistic effect, and a ratio of <1 indicates a less than additive effect.

Results

Table 6 of Figure 14 summarizes the food consumption, tumor incidence, tumor burden and the incidence of SCC in the buccal pouch of hamsters in experimental and control groups. At the end of 14 weeks, the tumor incidence in group 1 was 100 per cent. These tumors were exophytic and well defined with a mean tumor burden of 172.97 mm³. Although administration of bLF and Polyphenon-B alone and in combination (groups 2-4) significantly reduced the tumor incidence, tumor burden as well as the incidence of SCC, the combination was more effective than single agents. Furthermore, the combination index ratio of 1.46 for bLF and Polyphenon-B combination for tumor incidence indicates that the combination has a synergistic effect in inhibiting HBP carcinogenesis. No tumors were observed in groups 5-8. The amount of diet consumed in groups 1 to 8 was not significantly different.

Figure 15(A) and Table 7 of Figure 16 show the immunohistochemical analysis of cytokeratins in different groups. In DMBA painted animals (group 1) the expression of cytokeratins was significantly higher than in control animals. Furthermore, we observed that infiltrating carcinoma cells showed strong expression of cytokeratins in group 1 animals. The combination of bLF and P-B decreased cytokeratin expression more significantly than either agent alone. Administration of chemopreventive agents alone (groups 5-7) did not significantly influence the expression of cytokeratins compared to control (group 8)

Figure 15(B) and Table 7 of Figure 16 depict the irnmunohistochemical analysis of PCNA labeling index in the hamster buccal pouch of control and experimental animals. Topical application of DMBA (group 1) significantly increased the mean PCNA labeling index compared to control (group 8). Coadministration of bLF and P-B more significantly reduced the PCNA labeling index than either agent alone compared to group 1. Administration of bLF and P-B alone and in combination (groups 5 to 7) did not significantly affect the PCNA labeling index compared to untreated control (group 8).

The results of immunohistochemical analysis of mutant p53 protein expression and representative photomicrographs of p53 immunostaining are shown in Table 7 of Figure 15 (C). In DMBA painted animals, p53 expression was significantly increased compared to control (group 8). Combined administration of bLF and P-B reduced the expression of p53 more significantly than either agent alone. In animals administered chemopreventive agents alone (groups 5-7), the expression of p53 was not significantly different from than in control.

Figure 17 shows the representative Western blot analysis of Bcl-2, Bax, NF-kB, p53, Fas and caspase 3 as well as the activity of caspase 3 in the buccal pouch of control and experimental animals. The expression of Bcl-2, Bax, NF-kB, p53, Fas, and caspase 3 was detected as bands of molecular weight 25,21,65,53,48 and 32 kDa respectively. The mean protein expression from control lysates was designated as 100% in the graph. Each bar represents the mean protein expression ± SD of 10 determinations per treatment. Topical application of DMBA significantly increased the Bcl-2/Bax ratio as well as expression of p53 and NF-kB, and decreased Fas and caspase 3 expression and caspase 3 activity compared to control (group 1). Coadministration of bLF and P-B decreased the Bcl-2/Bax ratio, p53 and NF-kB expression and increased Fas and caspase 3 expression as well as the activity of caspase 3 more significantly than either agent alone. No significant changes in the expression of these proteins were observed in groups 5-7 compared to control animals.

Therefore, it should be appreciated that combined administration of bLF and a black tea polyphenol P-B was more effective than either agent alone in suppressing the development of DMBA-induced HBP carcinomas as revealed by decreased tumor incidence. This was associated with substantially decreased cell proliferation and significantly enhanced apoptosis as evidenced by downregulation of PCNA, NF-kB, ρ53 and Bcl-2 and upregulation of Bax, Fas and caspase 3 expression. The antiproliferative and apoptosis inducing effects of bLF and P-B could potentially mitigate the carcinogenic effects of DMBA, thereby decreasing the infiltrative potential of HBP carcinomas as reflected by cytokeratin expression.

The combinatorial chemopreventive effect of bLF and P-B reflects the antiproliferative and apoptosis-inducing effect of the individual agents. bLF has been reported to downregulate PCNA expression in 4-nitroquinoline 1 -oxide-induced tongue carcinogenesis. The inhibitory effect of bLF on azoxymethane induced colon carcinogenesis was found to be mediated by induction of the death receptor Fas as well as Bid and Bax, proapoptotic members of the Bcl-2 family. Tea polyphenols have been demonstrated to inhibit cell proliferation detected by in situ Brdu incorporation, PCNA downregulation and Gl phase arrest.

The results presented herein clearly demonstrate that combination of bLF with various tea polyphenols, and particularly black tea polyphenols act as a suppressing agent by inhibiting cell proliferation and inducing apoptosis. An interesting finding is the differential sensitivities of HBP carcinomas and normal cells to growth control and apoptosis. Several in vitro studies also demonstrated that tumor cells are more sensitive to the antiproliferative and apoptotic effects of tea polyphenols than normal cells. These findings support the fact that dietary agents that drive tumor cells to undergo apoptosis but direct normal cells toward a survival pathway are ideal chemopreventives. On the basis of these observations, our data suggest that a combination of bLF and P-B may have immense potential in human oral cancer prevention strategies.

We further evaluated the effect of a combination of lactoferrin and various black tea polyphenols (Polyphenon B (P-B)) on the cellular redox status and modulation of carcinogen metabolizing enzymes in 7,12-dimethylbenz[a]anthracene induced hamster buccal pouch (HBP) carcinogenesis. Chemicals

DMBA was purchased from Sigma Chemical Company, St. Louis, MO, USA. bLF (lot No. 020119) of purity 96.2% was obtained from Morinaga Milk Industry Co., Ltd, Tokyo, Japan. Black tea polyphenols (Polyphenon-B) were kindly provided by Mitsui Norm Co., Ltd., Tokyo, Japan. Polyphenon-B is a mixture of epicatechin (0.4%), epigallocatechin- 3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallocatechin-3-gallate (0.2%), free theaflavins (0.32%), theaflavinmonogallate-A (0.14%), theaflavinmonogallate-B (0.15%), theaflavindigallate (0.21%), tannin (35.6%) and caffeine (4.9%). All other reagents used were of analytical grade.

Animals and Diets

The experiment was carried out with male Syrian hamsters aged 8-10 weeks weighing 100-11Og obtained from the Central Animal House, Annamalai University, India. The animals housed five to a polypropylene cage were provided food and water ad libitum and maintained under controlled conditions of temperature and humidity with an alternating light/dark cycle. Animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India and approved by the ethical committee, Annamalai University. Experimental diets were prepared every day by mixing chemopreventive agents alone and in combination to a preweighed standard pellet diet (Mysore Snack Feed Ltd, Mysore, India). The diet was replenished everyday and the food consumption was recorded.

Treatment Schedule

The animals were randomized into experimental and control groups and divided into 8 groups of 20 animals each. In group 1, the right buccal pouches of hamsters were painted three times per week with a 0.5 per cent solution of DMBA in liquid paraffin with a number 4 brush. Each application leaves 0.4 mg. Hamsters in group 1 received no further treatment. In Groups 2-4, the right buccal pouches painted with DMBA, as in group 1, received in addition, basal diet containing 0.2 per cent bLF, 0.05 per cent Polyphenon-B and a diet containing combination of 0.2 per cent bLF and 0.05 per cent Polyphenon-B respectively. Animals in groups 5 through 7 were administered bLF, Polyphenon-B alone and in combination respectively. Group 8 animals served as control. The experiment was terminated at 14 weeks and all animals were killed by cervical dislocation after an overnight fast. Before an animal was killed, the right pouch was grossly inspected to evaluate premalignant lesions or tumor development and photographed. The tumor burden was calculated by multiplying the mean tumor volume (4/3 πr3) (r= ¹A tumor diameter in mm) with the mean number of tumors. The buccal pouch and liver tissues were subdivided and variously processed for distribution to each experiment.

Micronucleus Test

The bone marrow micronucleus test was carried out according to Schmid for evaluating chromosomal damage in experimental animals. A total of 2500 polychromatic erythrocytes were scored per animal from a single slide to determine the frequency of micronucleated polychromatic erythrocytes. All the slides were scored by the same observer.

Biochemical Assays

Fresh homogenized tissues were used for biochemical estimations. Cytochrome P450 was assayed by the method of Omura and Sato by using the carbon monoxide difference spectrum between 400 nm to 500 nm using an absorption coefficient of 91 cm² M-Im-I. Glutathione S-transferase (GST) activity was determined as described by Habig et al. by following the increase in absorbance at 340 nm using l-chloro-2,4-dinitrobenzene as substrate. The activity of DT-diaphorase (DTD) was assayed as described by Ernster using NADPH as the electron donor and 2,6-dichlorophenol-indophenol as the electron acceptor. Lipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances (TBARS), conjugated dienes and lipid hydroperoxides. TBARS were assayed in tissues by the method described by Ohkawa et al., lipid hydroperoxides by the method of Jiang et al. and conjugated dienes by the method of Rao and Recknagel. Reduced glutathione (GSH) was determined by the method of Anderson based on the development of yellow colour when 5,5'-dithiobis (2-nitrobenzoic acid) is added to compounds containing sulfhydryl groups. Oxidised glutathione (GSSG) was estimated following oxidation of NADPH by glutathione reductase at 340 nm based on the method of Anderson. Glutathione peroxidase (GPx) activity was assayed by following the utilization of hydrogen peroxide according to the method of Rotruck et al. The protein content was estimated by the method of Lowry et al. Statistical Analysis

The data are expressed as mean ± SD. Body weights were analysed using Student's t test. The X2 -test combined with Yates' correction was used to analyze the tumor incidence. Statistical analysis on the data for tumor burden, tumor multiplicity, incidence of bone marrow micronuclei and biochemical assays were analysed using analysis of variance (ANOVA) and the group means were compared by the Tukey-Kramer test. The results were considered statistically significant if the p value was <0.05. The nature of interaction between the combined effects of bLF and Polyphenon-B was evaluated as described by Yokoyama et al. In brief, the expected value of combined effect between treatment 1 (bLF) and treatment 2 (Polyphenon-B) is calculated as [(observed treatment 1 value)/(control value)] x [(observed treatment 2 value)/(control value)]; and the combination index is calculated as the ratio of (expected value)/(observed value). A ratio of >1 indicates a synergistic effect, and a ratio of <1 indicates a less than additive effect.

Macroscopic And Microscopic Observations

Table 8 of Figure 18 shows the mean body weights, food consumption, and the frequency of bone marrow micronuclei in different groups. Topical application of DMBA for 14 weeks significantly decreased the mean body weight of group 1 animals compared to control (group 8). No significant differences in body weights were observed in groups 2 to 8. The amount of diet consumed in groups 1 to 8 was not significantly different. The frequency of bone marrow micronucleated polychromatic erythrocytes (MnPCEs) was significantly higher in DMBA painted animals (group 1) compared to control (group 8). Although dietary administration of bLF and Polyphenon-B alone (groups 2 and 3 respectively) significantly reduced the incidence of MnPCEs compared to group 1, the combination of bLF and Polyphenon-B (group 4) showed a greater inhibition (43.33 per cent) compared to other groups. Administration of bLF and polyphenon-B alone and in combination (groups 5 to 7) showed no significant change in the incidence of MnPCEs compared to control (group 8).

Table 9 of Figure 19 summarizes the rumor incidence, tumor multiplicity, mean tumor burden and histopathological changes in the buccal pouch of hamsters in experimental and control groups. The tumor incidence in group 1 animals was 100 per cent with a multiplicity of 1.55 tumors per hamster. These tumors were exophytic and well defined with a mean tumor burden of 172.97 mm . Administration of bLF and Polyphenon-B alone and in combination (groups 2-4) significantly reduced the tumor incidence, tumor multiplicity and tumor burden as well as pathological changes. Among the groups 1-4, the combination of bLF and Polyphenon-B (group 4) more significantly reduced these changes. Furthermore, the combination index ratio of 1.46 for bLF and Polyphenon-B combination for tumor incidence indicates that the combination may have a synergistic effect in inhibiting HBP carcinogenesis. No tumors were observed in groups 5-8.

Biochemical Findings

Figure 20 illustrates the influence of treatment with bLF and Polyphenon-B alone and in combination on the activities of phase I and II carcinogen metabolizing enzymes in the buccal pouch and liver. Topical application of DMBA (group 1) significantly increased enzyme activities in the pouch, whereas in the liver, the increase in cytochrome P450 was accompanied by a significant decrease in phase II enzyme activities compared to control (group 8) (p<0.001). Dietary administration of bLF and Polyphenon-B alone and in combination to DMBA painted animals significantly inhibited the activities of cytochrome P450 and increased the activities of phase II enzymes in the pouch and liver compared to group 1 (p<0.05, p<0.01 and p<0.001 for groups 2-4 animals respectively). Although administration of dietary agents alone and in combination (groups 5-7) did not induce any significant change in cytochrome P450, the activities of phase II enzymes were significantly increased compared to group 8.

Figure 21 depicts the effect of treatment with bLF and Polyphenon-B alone and in combination on DMBA-induced changes in lipid peroxidation in the buccal pouch and liver of hamsters. Topical application of DMBA for 14 weeks lowered lipid peroxidation in the buccal pouch, whereas in the liver, the extent of lipid peroxidation was increased compared to control (p<0.001). Dietary administration of bLF and Polyphenon-B alone and in combination significantly modulated DMBA-induced changes in lipid peroxidation in the buccal pouch and liver compared to group 1 (p<0.05, p<0.01 and p<0.001 for groups 2-4 animals respectively). Administration of dietary agents alone and in combination significantly reduced the extent of lipid peroxidation in the buccal pouch and liver of groups 5-7 animals respectively compared to group 1. GSH, GSSG, GSH/GSSG ratio and GPx activity in the pouch and liver of control and experimental hamsters are shown in Figure 22. In DMBA painted animals (group 1), the concentration of GSH and GSH/GSSG ratio and GPx activity were increased in the buccal pouch, whereas in the liver, all the antioxidants were significantly decreased compared to control (p<0.001). Treatment with bLF and Polyphenon-B alone and in combination significantly increased all the antioxidants in the pouch and liver compared to group 1 (p<0.05, p<0.01 and p<0.001 for groups 2-4 animals respectively). Administration of bLF and Polyphenon-B alone and in combination (groups 5- 7) significantly enhanced the antioxidant status in the buccal pouch and liver compared to control (group 8). Overall, the combination of bLF and Polyphenon-B was found to be more effective than either agent used alone in modulating carcinogen-metabolizing enzymes as well as lipid peroxidation and antioxidants.

As can be seen, topical application of DMBA to the HBP for 14 weeks resulted in well-developed SCC with very high mean tumour burden. This was accompanied by an imbalance in phase I and II xenobiotic-metabolizing enzymes, cellular redox status and increased frequency of bone marrow micronuclei. Remarkably, coadministration of bLF and black tea polyphenols (Polyphenon-B) significantly decreased the incidence of HBP carcinomas, associated with modified balance in phase I and II enzymes and modulation of the redox status in the pouch and liver. Such balance and modulation is thought to mitigate the mutagenic and carcinogenic effects of DMBA, thereby minimizing the frequency of bone marrow micronuclei.

the inventors further found that bLF and Polyphenon-B function as dual-acting agents by suppressing phase I enzymes and enhancing the activities of phase II enzymes. Dual- acting agents are recognized to be more promising as cancer chemopreventive agents because they simultaneously inhibit metabolic activation of carcinogens while promoting detoxification and excretion. The chemopreventive potential of bLF and Polyphenon-B combination may also be attributed to its antioxidant properties. In the buccal pouch, bLF and Polyphenon-B reversed the susceptibility to lipid peroxidation whilst simultaneously increasing GSH/GSSG ratio and GPx activity, whereas in the liver, the extent of lipid peroxidation was reduced with elevation of antioxidant defense system. Thus the differential oxidative events induced in the tumor and host liver by bLF and Polyphenon-B may presumably reduce cell proliferation and block tumor development in the HBP. The results of the present study suggest that combined administration of bLF and Polyphenon-B is more effective in inhibiting HBP carcinogenesis and modulating carcinogen metabolizing enzymes and the redox status than either agent used alone. These findings further suggest that bLF and Polyphenon-B combination can act synergistically in inhibiting HBP carcinogenesis. (III) Effects of Green Tea Polyphenol-Lactoferrin Combinations

We further evaluated the antiproliferative and apoptosis inducing effects of bovine lactoferrin in combination with various green tea polyphenols (Polyphenon E) and black tea polyphenols (Polyphenon E) on human tongue squamous carcinoma (CAL-27) and normal human gingival fibroblast (HGF) cells.

Chemicals

bLF (lot No. 020119) of purity >96.2% was obtained from Morinaga Milk Industry Co., Ltd, Tokyo, Japan. The iron content of bLF was 18mg/100g. Green tea polyphenols (Polyphenon-E:P-E) and black tea polyphenols (Polyphenon-B:P-B) were kindly provided by Mitsui Norin Co., Ltd., Tokyo, Japan. The composition of Polyphenon-E and Polyphenon-B is same as described previously.16 Polyphenon-E (P-E) is a mixture of epigallocatechin-3- gallate (64.6%), epigallocatechin (4.3%), epicatechin (9.4%), epicatechin-3-gallate (6.4%), gallocatechin-3-gallate (3.5%), catechin-3-gallate (0.2%), gallactocatechin (0.2%), catechins (1.1%) and caffeine (0.7%). Polyphenon-B (P-B) has the following composition: epicatechin (0.4%), epigallocatechin-3-gallate (1.4%), epicatechin-3-gallate (0.1%), gallocatechin-3- gallate (0.2%), free theaflavins (0.32%), theaflavinmonogallate-A (0.14%), theaflavinmonogallate-B (0.15%), theaflavindigallate (0.21%), tannin (35.6%) and caffeine (4.9%). AU other reagents used were of analytical grade. Stock solutions of bLF and P-E were prepared in phosphate buffered saline (PBS). P-B was dissolved in PBS containing 0.5% dimethyl sulfoxide (DMSO). The stock solutions were then diluted with the medium prior to use to obtain the desired concentration. The final concentration of DMSO in the medium was less than 0.01 per cent that proved to have no detectable effect on cell growth. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO. Dithiothreitol (DTT), 3,3-diaminobenzidine tetrahydrochloride (DAB), 2',7'-dichlorofluorescein diacetate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT), propidium iodide, proteinase K, phenylmethanesulfonyl fluoride (PMSF), rhodamine 123, and RNase A were purchased from Sigma Chemical Company, St. Louis, MO, USA.Cell lines and cell cultures Normal human gingival fibroblast (HGF) and human tongue squamous cell carcinoma (CAL-27) cell lines generously provided by Dr.D.A.Tipton, University of Tennessee, College of Dentistry, Memphis, TN, USA were used in this study. The cells were grown in DMEM supplemented with 10% FBS, 50U/ml penicillin G, and 50μg/ml streptomycin sulphate. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. Exponentially growing cells were used for all the experiments.

Cytotoxicity Assay

Cytotoxicity was assessed by the MTT assay based on the reduction of MTT by mitochondrial dehydrogenases of viable cells to a purple formazon product.17 Briefly, cells were diluted in growth medium and seeded in 24- well plates (5x104 cells/well). After overnight growth, the growth medium was replaced with exposure medium (DMEM without FBS) containing indicated doses of bLF, P-E, P-B alone and a combination of bLF and P-E or P-B. After 24h, the cells in each well were washed with 200μl of PBS, and incubated with lOOμl of 500μg/ml MTT in PBS at 37°C for 3 h. The MTT-formazon product dissolved in 200 μl of DMSO was estimated by measuring the absorbance at 570nm in an ELISA plate reader. Cell survival was expressed as percentage of viable cells of treated samples to control samples. All the dietary agents were tested in triplicates and the experiments were repeated at least three times.

Nuclear Morphology

CAL-27 cells were plated at a density of 5x104 cells/well into 6 well chamber slides. After 80% confluence, CAL-27 cells were treated with dietary agents alone and in combination for 24 h. The cells were then washed with PBS, fixed in methanol: acetic acid (3:1, v/v) for 10 minutes and stained with 50μg/ml propidium iodide for 20 minutes. Nuclear morphology of apoptotic cells with condensed/fragmented nuclei was examined under a fluorescent microscope and at least 1x103 cells were counted for assessing apoptotic cell death.

Cell Cycle Analysis

Cell cycle distribution and measurement of the percentage of apoptotic cells were performed by flow cytometry. After treatment, floating cells in the medium were combined with attached cells harvested by trypsinisation. Cells were then washed with cold PBS and fixed in 80% ethanol in PBS at -20°C. After 12 h, fixed cells were pelleted and stained with propidium iodide (50μg/ml) in the presence of RNase A (20μg/ml) for 30 min at 37°C, and about 10⁴ events were analysed on a Becton Dickinson FACScan flow cytometer. Cell cycle histograms were analysed using Cell Quest software. Apoptotic cells were distinguished form non-apoptotic intact cells by their decreased DNA content as determined by their lower propidium iodide staining intensity appearing in the area below subGO/Gl phase.

Determination OfROS Generation

To assess the generation of intracellular ROS, the oxidation-sensitive fluorescent probe DCFH-DA was used. Briefly, after treatment, CAL-27 cells were harvested and suspended in 0.5ml PBS containing lOμM DCFH-DA for 15 minutes at 37°C in dark. DCFH- DA was taken up by cells and deacetylated by cellular esterase to form a nonfluorescent product DCFH, which was converted to a green fluorescent product DCF by intracellular ROS produced by treated CAL-27 cells. The intensity of DCF fluorescence was measured by flow cytometry with excitation and emission settings of 488 and 530 nm respectively.12 Totally 104 events were counted and the histograms were analysed using Cell Quest software and compared with histograms of control untreated cells.

Determination Of Mitochondrial Transmembrane Potential

The changes in mitochondrial transmembrane potential (Δψm) were measured by uptake of the mitochondrial specific lipophilic cation dye rhodamine 123.20 After treatment, CAL-27 cells were pelleted by centrifugation for 10 min at room temperature and washed with PBS. The pelleted cells were incubated with ImI of exposure medium containing lOμg/ml rhodamine 123 for 30 min at room temperature in dark, washed and resuspended in PBS. The samples (104 events) were then immediately subjected to flow cytometric analysis at an excitation wavelength of 488nm and an emission wavelength of 545nm. Histograms were anlaysed using Cell Quest software and compared with histograms of control untreated cells.

Immunofluorescence

CAL-27 cells cultured to about 80% confluence in 6 well chamber slides were exposed for 24 h to dietary agents alone and in combination. Following treatment, cells were fixed in pre-chilled acetone at 4°C for 5 min. The fixed cells were permeabilised with 0.1% Triton X-IOO in PBS and incubated with 1:1000 dilution of anti-Bcl-2 and Bax antibody (Dako, Carpinteria, CA, USA) at 4oC overnight. The proteins were then detected by incubating with fluorescein isothiocyanate (FITC)-conjugated secondary anti-mouse IgG antibody (Dako, Carpinteria, CA, USA) and visualized using a fluorescent microscope. Western Blotting

Following treatment with the dietary agents, CAL-27 cells were washed twice with ice-cold PBS, lysed in lysis buffer (5OmM Tris-HCl, pH 8.0, 5mM EDTA, 15OmM sodium chloride, 0.5 % Nonidet P-40, 0.5 niM PMSF and 0.5 mM DTT) for 30 min at 4⁰C, and the supernatant was collected by centrifuging at 12,500 x g for 20min. About 50μg of total protein from the supernatant as determined by Bradford's protein estimation kit, was resolved on 10% SDS-PAGE, and then transferred to a nitrocellulose membrane using a semi-dry transfer system (BIORAD).21 The membranes were incubated in Tris buffered saline (TBS) (150mM/L sodium chloride, 50 mM/L Tris, pH 7.4) containing 5% non-fat dry milk for Ih to block non-specific binding sites. The blocks were then incubated with 1:1000 dilution of anti- Bcl-2 and Bax (NeoMarkers, USA) overnight. The blocks were extensively washed with TBS containing 0.1% Tween-20 and the proteins were detected by incubating with corresponding horse-radish peroxidase-conjugated secondary antibodies (1:2000) for 60-90min at room temperature. After extensive washes in TBS containing 0.1% Tween 20, the transferred proteins were visualized using DAB. Densitometry was performed on an IISP flat-bed scanner and quantified with Total Lab 1.11 software.

Colorimetric Estimation Of Caspase-3 Activity

Caspase-3 activity was assayed using CASP-3-C colorimetric kit (Sigma Chemical Company, St. Louis, MO, USA). After treatment, CAL-27 cells were lysed in lysis buffer containing 250mM/l HEPES (pH 7.4), 25 mM/L CHAPS and 25mM/L DTT. The supernatant was used as an enzyme source. The caspase-3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-nitroanilide (Ac-DEVD-pNA) by caspase-3 that results in release of p-nitroaniline (pNA) moiety. The concentration of pNA released from the substrate was calculated from the absorbance values at 405nm or from a calibration curve prepared with defined pNA solutions.

Statistical Analysis

Cytotoxicity data are presented as mean percentages of control ± S. D and linear regression analysis was used to calculate the IC50 values. Statistical analysis on the data for cytotoxicity of tea polyphenols alone and in combination with bLF on CAL-27 and HGF cells was done using analysis of variance (ANOVA). The nature of interaction between the combined effects of tea polyphenols and bLF was evaluated as described by Yokoyama et al.22 Synergism was calculated from the ratio of expected value (E)/observed value (O) of bLF and tea polyphenols combination treatment; a ratio of > 1 indicates a synergistic effect; E value of bLF and tea polyphenols = [(O value of bLF) / (O value of control cells)] x [(O value of tea polyphenols) / (O value of control cells)] x (O value of control cells). The data for nuclear morphology, cell cycle analysis, Bcl-2/Bax ratio and caspase 3 activity were analysed by Student's t test. The results were considered statistically significant if the p value was <0.05.

Results

Cytotoxicity assay: We first examined the inhibitory effects of different concentrations of P-E, P-B and bLF on the growth of CAL-27 and HGF cells (Figure 23). Both P-E and P-B showed a dose-dependent inhibition of growth of CAL-27 cells with IC50 values of 20 and 40μg/ml respectively. However, HGF cells were more resistant to the growth inhibitory effects of P-E and P-B with IC50 values of 70 and 120μg/ml respectively. Treatment with bLF did not induce any cytotoxic effects either in CAL-27 or in HGF cells. We next examined the growth inhibitory effects of tea polyphenols in combination with bLF on CAL-27 and HGF cells. We chose the IC50 value of P-E and P-B on CAL-27 cells and tested this in combination with increasing concentration of bLF. Figure 24A shows U-shaped growth inhibition of CAL-27 cells by a combination of P-E with increasing concentration of bLF. Significant synergistic effects were observed with P-E (20μg/ml)+bLF (40μg/ml) combination at a ratio of 1:2. HGF cells appeared to be less susceptible to growth inhibition than CAL-27 cells indicating the preferential growth inhibition of CAL-27 cells by P-E and bLF combination. Figure 24B illustrates the effect of treatment with a combination of P-B and bLF on the growth of CAL-27 and HGF cells. Cotreatment with P-B and bLF significantly reduced the susceptibility of CAL-27 cells to growth inhibition compared to P-B alone. These results suggest that bLF suppresses the anticancer effects of P-B. Overall, the results of MTT assay suggest that dietary agents preferentially inhibit the growth of CAL-27 cells compared to normal HGF cells and the order of their cytotoxic effect was P-E and bLF combination (1:2 ratio)>P-E>P-B. Since the dietary agents preferentially inhibited the growth of CAL-27 cells and addition of bLF suppressed the anticancer effects of P-B, further studies were conducted only in CAL-27 cells by incubating with P-E (20μg/ml), P-B (40μg/ml) and bLF (40μg/ml) alone and a combination of P-E (20μg/ml) with bLF (40μg/ml) to explore the mode of cell death. Induction of apoptosis in CAL-27 cells by dietary agents: To determine whether the inhibition of cell growth by dietary agents is caused by apoptosis, nuclear morphology was observed using the fluorescent DNA-binding agent propidium iodide. Incubation of CAL-27 cells with the dietary agents for 24 h significantly increased the number of apoptotic cells compared to control as evidenced by nuclear fragmentation and condensation (Figure 25). Treatment of CAL-27 cells with P-E (20 μg/ml) and P-B (40 μg/ml) alone significantly increased the number of apoptotic cells compared to control (p<0.01 and p<0.05 respectively). Although bLF alone did not induce apoptosis, cotreatment with P-E (20 μg/ml) and bLF (40 μg/ml) significantly increased the number of apoptotic nuclei to 60.24% (p<0.005 and p<0.05 compared to control and P-E alone treated cells respectively). Specifically, 40μg/ml bLF enhanced the apoptosis inducing potential of 20 μg/ml P-E by -1.9 fold. The order of apoptosis inducing potential of dietary agents was P-E and bLF (1 :2) > P-E > P-B.

Effect of dietary agents on the cell cycle control of CAL-27 cells: To investigate whether the dietary agents have a role on cell cycle regulation, we analyzed the changes in cell cycle profiles by using fluorescent activated cell sorter. As shown in Figure 26, incubation of CAL-27 cells with dietary agents for 24 h, significantly increased the proportion of cells with a reduced DNA content (subGO/Gl or AO peak) indicative of apoptosis with loss of cells in Gl phase. Loss of DNA, a hallmark of apoptosis, occurs as a result of diffusion of degraded DNA out of the cells after endonuclease cleavage, and after staining with propidium iodide, these cells would have taken up less stain and appear in subG0/Gl or AO peak i.e. to the left of the G0/Glpeak. Incubation of CAL-27 cells with P-E and P-B alone significantly increased the proportion of cells with a reduced DNA content from 8.36 per cent (control) to 35.99 per cent (P-E) and 24.01 per cent (P-B). Although bLF alone did not significantly alter the percentage of subG0/Gl cells, cotreatment with 20μg/ml P-E and 40μg/ml bLF significantly increased the percentage of subG0/Gl cells to 72.86% (p<0.005 and p<0.01 compared to control and P-E alone treated cells respectively). Specifically, 40μg/ml bLF enhanced the apoptosis inducing potential of 20 μg/ml P-E by ~2 fold. The order of apoptosis inducing potential of dietary agents was the same

as for nuclear morphology analysis.

Effect of dietary agents on intracellular ROS generation: To investigate whether ROS is involved in mediating apoptosis induced by the dietary agents we measured the intracellular generation of ROS using the fluorescent probe DCFH-DA. Treatment with dietary agents significantly increased ROS generation in CAL-27 treated cells compared to untreated control, and the order of ROS generation was P-E+bLF>P-E>P-B (Figure 27A). The mean of DCF fluorescence increased from Ma=381 (control) to Mb=938 (P-E treated), Mc=1229 (P-E and bLF, 1:2 ratio) and Md=649 (P-B treated). Exposure of CAL-27 cells to bLF alone did not induce ROS generation (data not shown).

Effect of dietary agents on mitochondrial transmembrane potential (Δψm): The increasing evidence that changes in Δψm are linked to apoptosis led us to examine the influence of dietary agents alone and in combination on Δψm of CAL-27 cells by flow cytometry using the fluorescent probe rhodamine 123. Rhodamine 123, a cationic dye, is selectively taken up by mitochondria to an extent that is directly proportional to the Δψm. As illustrated in Figure 27B, CAL-27 cells exposed to tea polyphenols for 24 h, showed a loss of Δψm as revealed by reduced rhodamine 123 fluorescence intensity compared to control. The mean rhodamine 123 fluorescence declined from Ma=4636 (control) to Mb=2456.12 (P-E treated) and Md=2659.88 (P-B treated). Although bLF alone did not show any significant alteration in Δψm (data not shown) treatment with a combination of P-E and bLF showed a further sharp decline in rhodamine 123 fluorescence intensity to 2098.75 compared to control and P-E alone treated cells.

Effect of dietary agents on Bcl-2/Bax ratio: We examined the effects of dietary agents on the expression of Bcl-2 and Bax that play a key role in regulating apoptosis by controlling Δψm using immunofluorescence (data not shown) and Western blotting. Incubation of CAL- 27 cells with P-E, P-B and a combination of P-E and bLF (1:2 ratio) significantly decreased the Bcl-2/Bax protein expression compared to untreated control and the order of Bcl-2/Bax decreasing potential of dietary agents was P-E+bLF>P-E>P-B (Figure 28A). Effect of dietary agents on caspase 3 activity: The assay of caspase 3 in CAL-27 cells treated with dietary agents revealed that apoptosis induction was mediated through activation of caspase 3 activity (Figure 28B). Incubation of CAL-27 cells with P-E, P-B, and a combination of P-E and bLF (1:2 ratio) significantly increased caspase 3 activity compared to control. While bLF did not exhibit any effect on caspase 3 activity, cotreatment of bLF with P-E increased enzyme activity by ~1.46 fold.

These results strongly suggest that tea polyphenols preferentially inhibit the growth of human tongue squamous carcinoma (CAL-27) cells in a dose dependent manner. The present data further indicate that P-E is more effective than P-B in inhibiting the growth of CAL-27 cells. Furthermore, a combination of P-E andbLF (1:2 ratio) exerts significant synergistic effect in inhibiting tumor growth as reflected by a U-shaped growth curve, whereas cotreatment with bLF suppressed the anticarcinogenic effects of P-B. Overall, the preferential inhibition of growth of CAL-27 cells and the order of the antiproliferative effects was P- E+bLF(l:2)>P-E>P-B. The greater efficacy of P-E in inhibiting growth of CAL-27 cells apparently reflects its higher concentration of EGCG. The present results also demonstrate that green and black tea polyphenols inhibit the growth of CAL-27 cells by inducing apoptosis as revealed by characteristic changes in nuclear morphology and Ao peak. There is increasing evidence that apoptosis induced by chemopreventive or chemotherapeutic agents is associated with perturbation of a specific phase of the cell cycle. As shown in Figure 4, incubation of CAL-27 cells with tea polyphenols for 24h resulted in loss of cells in Go/Gl phase with concomitant increase in appearance of apoptotic cells (Ao peak), suggesting that CAL-27 cells arrested in G0/G1 phase by tea polyphenols are preferentially undergoing apoptosis. While bLF alone did not induce apoptosis of CAL-27 cells, cotreatment of P-E with bLF enhanced the apoptosis-inducing potential of P-E by ~2 fold. The order of apoptosis inducing potential of dietary agents was same as that of the growth inhibitory effects i.e. P- E+bLF(l:2)>P-E>P-B.

Mitochondria, which play a pivotal role in apoptosis, are major sites of ROS generation. Excessive generation of ROS can lead to opening of the mitochondrial permeability transition pore with decline in Δψm and consequent release of cytochrome c from the intermembrane space into the cytosol culminating in activation of the caspase cascade and apoptotic cell death. The results of the present study demonstrate that incubation of CAL-27 cells with P-E, P-B and P-E +bLF (1 :2 ratio) increased ROS generation, which in turn triggered apoptosis by disrupting mitochondrial function as revealed by decrease in Δψm, and activation of caspase-3. EGCG has been reported to induce apoptosis in Jurkat cells by generation of ROS. Several studies have provided evidence for the involvement of ROS, dissipation of Δψm and caspase activity during apoptosis induced by EGCG and tea polyphenols. Taken together, these results suggest that tea polyphenols induce apoptosis via ROS-induced mitochondrial perturbation.

In summary, the present results demonstrate that tea polyphenols exert growth inhibitory effects against CAL-27 cells that is mediated through apoptosis. The data indicate a key role for ROS and Bcl-2/Bax in mitochondrial mediated apoptosis by dissipating Δψm and activating caspase-3. The study also emphasizes the greater efficacy of P-E both alone and in combination with bLF. Since green tea polyphenols have already entered clinical trials in patients at high risk for liver and prostate cancers, it would be worthwhile to design similar trials in patients with oral premalignant lesions to evaluate the chemopreventive efficacy of P-E.

Thus, specific embodiments and applications of tea polyphenols have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Claims

CLAIMSWhat is claimed is:

1. A method of improving treatment or chemoprevention of oral cancer using either oral administration of a tea polyphenol composition or oral administration of a lactoferrin, comprising:

ascertaining that oral administration of the tea polyphenol composition or oral administration of the lactoferrin reduces oral cancer with a first efficiency; and

providing information that combined oral administration of the tea polyphenol composition and the lactoferrin reduces oral cancer with a second efficiency that is greater than the first efficiency.

2. The method of claim 1 wherein the tea polyphenol composition comprises a mixture of a plurality of catechins, optionally polymerized to polymerized catechins.

3. The method of claim 1 wherein the tea polyphenol composition comprises a mixture of a plurality of catechins obtained from green tea.

4. The method of claim 1 wherein the lactoferrin is bovine lactoferrin and wherein the tea polyphenol composition comprises a mixture of a plurality of catechins, optionally polymerized to polymerized catechins, and wherein the combined lactoferrin and catechins are optionally formulated as a liquid product, a capsule, a tablet, or a powder.

5. The method of claim 1 wherein a tea polyphenol composition to lactoferrin weight ratio is between 1:1 and 1:3 in the combined oral administration.

6. The method of claim 5 wherein the ratio is synergistic.

7. The method of claim 1 wherein oral administration is coadministration in a single dosage unit form.

8. A nutraceutical or pharmaceutical product comprising a combination of a tea polyphenol composition and lactoferrin in an amount effective that reduces oral cancer at an efficiency that is greater than an efficiency achieved by oral administration of the product with either the tea polyphenol composition or the lactoferrin alone.

9. The product of claim 8 wherein the polyphenol composition comprises a mixture of a plurality of catechins obtained from at least one of black tea and green tea, and wherein the product is optionally formulated as a liquid product, a capsule, a tablet, or a powder.

10. The product of claim 8 wherein the polyphenol composition comprises at least one synthetic catechin.

11. The product of claim 8 wherein the polyphenol composition is present in an amount of between 100 mg and 600 mg per dosage unit, and wherein the lactoferrin is present in an amount of between 200 mg and 1200 mg per dosage unit.

12. The product of claim 8 wherein the product is a non-liquid nutraceutical product that is formulated to include both the polyphenol composition and the lactoferrin.

13. The product of claim 8 wherein the product is a nutraceutical product, and wherein at least one of the polyphenol composition and the lactoferrin is in a liquid carrier.

14. The product of claim 8 wherein the greater efficiency is a synergistic efficiency.

15. Use of a combination of a tea polyphenol composition and a lactoferrin in the production of a nutraceutical or pharmaceutical product for treatment or chemoprevention of oral cancer.

16. The use of claim 15 wherein the tea polyphenol composition and the lactoferrin are present in a weight ratio of between 1:1 and 1:3.

17. The use of claim 16 wherein the weight ratio is a synergistic weight ratio.

18. The use of claim 15 wherein the tea polyphenol composition comprises a mixture of a plurality of catechins obtained from at least one of black tea and green tea.

19. The use of claim 15 wherein the combination is formulated into a single dosage unit, optionally formulated as a liquid product, a capsule, a tablet, or a powder.

20. The use of claim 15 wherein the product is a pharmaceutical product, optionally formulated as a liquid product, a capsule, a tablet, or a powder.

21. A method of preventing oral cancer, comprising administering a combination of a tea polyphenol and lactoferrin.

22. The method of claim 12 wherein the step of administering comprises oral administration, and wherein the combination is formulated into a single dosage unit.

23. The method of claim 12 wherein the tea polyphenol comprises a mixture of a plurality of catechins obtained from at least one of black tea and green tea.

24. The method of claim 12 wherein the combination is formulated in a product selected from the group consisting of a liquid nutritional product, a capsule, a tablet, and a powdered formulation.