Inhibition of cellular transformation by berry extracts.

Recent studies have examined and demonstrated the potential cancer chemopreventive activity of freeze-dried berries including strawberries and black raspberries. Although ellagic acid, an abundant component in these berries, has been shown to inhibit carcinogenesis both in vivo and in vitro, several studies have reported that other compounds in the berries may also contribute to the observed inhibitory effect. In the present study, freeze-dried strawberries (Fragara ananassa, FA) or black raspberries (Rubus ursinus, RU) were extracted, partitioned and chromatographed into several fractions (FA-F001, FA-F003, FA-F004, FA-F005, FA-DM, FA-ME from strawberries and RU-F001, RU-F003, RU-F004, RU-F005, RU-DM, RU-ME from black raspberries). These extracts, along with ellagic acid, were analyzed for anti-transformation activity in the Syrian hamster embryo (SHE) cell transformation model. None of the extracts nor ellagic acid by themselves produced an increase in morphological transformation. For assessment of chemopreventive activity, SHE cells were treated with each agent and benzo[a]pyrene (B[a]P) for 7 days. Ellagic acid, FA-ME and RU-ME fractions produced a dose-dependent decrease in transformation compared with B[a]P treatment only, while other fractions failed to induce a significant decrease. Ellagic acid, FA-ME and RU-ME were further examined using a 24 h co-treatment with B[a]P or a 6 day treatment following 24 h with B[a]P. Ellagic acid showed inhibitory ability in both protocols. FA-ME and RU-ME significantly reduced B[a]P-induced transformation only when co-treated with B[a]P for 24 h. These results suggest that a methanol extract from strawberries and black raspberries may display chemopreventive activity. The possible mechanism by which these methanol fractions (FA-ME, RU-ME) inhibited cell transformation appear to involve interference of uptake, activation, detoxification of B[a]P and/or intervention of DNA binding and DNA repair.

[Studies on chemical constituents of Fragaria ananassa Duch].

OBJECTIVE: To study the chemical constituents from the fruit of Fragaria ananassa. METHOD: Using chromatographic methods to isolate compounds and chemical and spectral methods to elucidate their structures. RESULT: Three compounds, 9, 19-cyclolanost-24-en-3-ol(1), 14-methyl-stigmasta-7, 24(28)-dien-3-ol(2) and beta-sitosterol(3) were isolated from the freeze-dried powder. CONCLUSION: All of the compounds were obtained from this plant for the first time.

Chemoprevention with theaflavins of rat esophageal intraepithelial neoplasia quantitatively monitored by image tile analysis.

The objective of the study was to compare three methods of monitoring the inhibition by dietary theaflavins of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal intraepithelial neoplasia: the mean tile grade, measured by computer-assisted quantitative image tile analysis; tumor multiplicity; and mean tumor size. A "tile" is defined as a small portion of a microscopic image at x 40, 87 x 292 microm in size. The computer divided the image of esophageal intraepithelial neoplasia into a grid of contiguous tiles and measured four tissue features within each tile based on cytonuclear and tissue architectural changes used by pathologists to diagnose intraepithelial neoplasia. The tile grade is defined as the weighted sum of the four feature measurements within a tile, the weights being determined by Fisher linear discriminant analysis. The mean tile grade of 300 tiles is used to grade rat esophageal intraepithelial neoplasia. NMBA was given s.c., 0.5 mg/kg, three times a week for 5 weeks. Theaflavins were given in the drinking water at 360 ppm (low dose) and 1200 ppm (high dose) throughout the experiment. In a given set of four groups of rats, one group received theaflavins alone, one NMBA alone, one NMBA plus low-dose theaflavins, and one NMBA plus high-dose theaflavins. One set of four groups, four rats/group, was sacrificed at the 15th week and another at the 20th week after starting NMBA; a final set with 15 rats/group was sacrificed at 25 weeks. At the 15th and 20th weeks, the mean tumor grade was the only variable that responded significantly (P < 0.01) to the low dose of dietary theaflavins. In fact, tumor multiplicity and mean tumor size sometimes showed enhancement at these doses. At the 25th week, when there were 15 instead of 4 rats/group, the mean tile grade, tumor multiplicity, and mean tumor size were all significantly (P < 0.01) decreased by both low and high doses of theaflavins. The mean tile grade is a more sensitive and reproducible variable than tumor multiplicity and mean tumor size in detecting the chemopreventive effects of theaflavins on intraepithelial neoplasia in the rat esophagus. This suggests that the mean tile grade may be a useful intermediate end point for use in human chemoprevention trials.

Inhaled isotretinoin (13-cis retinoic acid) is an effective lung cancer chemopreventive agent in A/J mice at low doses: a pilot study.

In previously treated head-and-neck cancer patients, p.o. administered isotretinoin (13-cis retinoic acid) reduced the occurrence of second aerodigestive tumors, including lung tumors, but side effects made chronic therapy problematic. We reasoned that inhaled isotretinoin might provide sufficient drug to the target cells for efficacy while avoiding systemic toxicity, and we proceeded with the pilot study reported here. Male A/J mice were given single i.p. doses of urethane, a common experimental lung carcinogen, or benzo[a]pyrene (BaP) or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), putative major carcinogens in tobacco smoke. The following day, exposures to isotretinoin aerosols for 45 min daily at 1.3, 20.7, or 481 microg/l were initiated. After 2 weeks, the high dose caused severe toxicity on the snout skin, necessitating a reduction of dose frequency to twice a week. As a precaution, the mid dose was reduced to three exposures per week. The weekly total deposited doses after the dose frequency reductions were calculated to be 0.24, 1.6, and 24.9 mg/kg for the low, mid, and high doses, of which 16% was estimated to be deposited in the lungs. The weekly deposited pulmonary drug doses were calculated to be 0.01, 0.07, and 1.1% of a previously reported ineffective oral dose in urethane-treated A/J mice. After 10-16 weeks, mice were sacrificed to count areas of pulmonary hyperplasia and adenomas. For all carcinogens, the mice exposed to the high isotretinoin dose showed reductions of tumor multiplicity ranging from 56 to 80% (P < 0.005). The mid dose was associated with reductions of tumor multiplicity by 67 and 88% (P < 0.005) in BaP- and NNK-treated mice, respectively, and was tolerated until approximately 12 weeks, when both these and the high-dose mice began losing weight. The low-dose mice had nonsignificant reductions of 30% (P < 0.13) and 16% (P < 0.30) for BaP- and NNK-treated mice, respectively without any evidence of side effects. For BaP- and NNK-treated mice, numbers of hyperplastic areas directly correlated to dose level and inversely to tumor number, suggesting arrested progression. Inhaled mid-dose isotretinoin caused up-regulation of lung tissue nuclear retinoic acid receptors (RARs) relative to vehicle-exposed mice, RARalpha (3.9-fold vehicle), RARbeta (3.3-fold), and RARgamma (3.7-fold), suggesting that these receptors may be useful biomarkers of retinoid activity in this system. The encouraging results from this pilot study suggest that inhaled isotretinoin merits evaluation in people at high risk for lung cancer.

Effects of theaflavins on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis.

The purpose of this experiment was to compare the inhibitory effects of the polyphenol fraction of black tea, theaflavins (TF), the polyphenol fraction of green tea, and (-)-epigallocatechin-3-gallate (EGCG) in the rat esophageal tumor model. The tea fractions were administered in the drinking water at concentrations of 360 and 1,200 ppm for two weeks before administration of the esophageal carcinogen N-nitrosomethylbenzylamine (NMBA). NMBA was administered subcutaneously in 10% dimethyl sulfoxide three times weekly for five weeks. Additional groups of rats received only vehicle and plain drinking water or vehicle and drinking water containing 1,200 ppm of each tea fraction. Twenty-five weeks after NMBA administration began, the experiment was terminated and esophagi were excised and scored for tumors. Rats that were not dosed with NMBA had no tumors. Rats treated with NMBA only had an esophageal tumor incidence of 100% and a multiplicity of 3.3 +/- 0.4 tumors/rat. The proportion of rats developing tumors was not significantly reduced by any of the four tea fractions at the concentrations tested. However, the 1,200 ppm concentrations of each tea fraction in the drinking water produced some reduction in esophageal tumor multiplicity, although only TF significantly reduced tumor multiplicity compared with rats treated with NMBA only. The rates of esophageal tumor formation were significantly reduced at 360 and 1,200 ppm by TF and EGCG.

Effects of dietary phenethyl isothiocyanate, ellagic acid, sulindac and calcium on the induction and progression of N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in rats.

The potential inhibitory effects of phenethyl isothiocyanate (PEITC), ellagic acid (EA), sulindac and supplemental dietary calcium (SDC) on N-nitrosomethylbenzylamine (NMBA)-induced esophageal carcinogenesis were evaluated in rats utilizing an abbreviated (5 week) NMBA treatment protocol which allowed administration of the putative inhibitors throughout the experiment (i.e. beginning 2 weeks prior to NMBA treatment) or following completion of NMBA dosing only. PEITC at 500 p.p.m. significantly inhibited tumor incidence and multiplicity when given before and during, but not following, NMBA treatment. Neither sulindac at 125 p.p.m. nor SDC (2% versus 0.5% in control diet) inhibited tumor development when given during or following NMBA treatment. EA, which was administered only following NMBA treatment, significantly reduced the incidence (66.7% versus 100% in NMBA controls), but not the multiplicity, of esophageal tumors at the high-dose (4000 p.p.m.) level. Together these findings indicate that: (i) PEITC selectively inhibits the induction but not the subsequent progression of NMBA-induced esophageal tumors; (ii) EA may repress esophageal tumor development when administered following NMBA treatment; (iii) at the doses administered, neither sulindac nor SDC possess significant inhibitory activity against NMBA-induced esophageal carcinogenesis in the rat.

Polyphenols as cancer chemopreventive agents.

This article summarizes available data on the chemopreventive efficacies of tea polyphenols, curcumin and ellagic acid in various model systems. Emphasis is placed upon the anticarcinogenic activity of these polyphenols and their proposed mechanism(s) of action. Tea is grown in about 30 countries and, next to water, is the most widely consumed beverage in the world. Tea is manufactured as either green, black, or oolong; black tea represents approximately 80% of tea products. Epidemiological studies, though inconclusive, suggest a protective effect of tea consumption on human cancer. Experimental studies of the antimutagenic and anticarcinogenic effects of tea have been conducted principally with green tea polyphenols (GTPs). GTPs exhibit antimutagenic activity in vitro, and they inhibit carcinogen-induced skin, lung, forestomach, esophagus, duodenum and colon tumors in rodents. In addition, GTPs inhibit TPA-induced skin tumor promotion in mice. Although several GTPs possess anticarcinogenic activity, the most active is (-)-epigallocatechin-3-gallate (EGCG), the major constituent in the GTP fraction. Several mechanisms appear to be responsible for the tumor-inhibitory properties of GTPs, including enhancement of antioxidant (glutathione peroxidase, catalase and quinone reductase) and phase II (glutathione-S-transferase) enzyme activities; inhibition of chemically induced lipid peroxidation; inhibition of irradiation- and TPA-induced epidermal ornithine decarboxylase (ODC) and cyclooxygenase activities; inhibition of protein kinase C and cellular proliferation; antiinflammatory activity; and enhancement of gap junction intercellular communication. Curcumin is the yellow coloring agent in the spice tumeric. It exhibits antimutagenic activity in the Ames Salmonella test and has anticarcinogenic activity, inhibiting chemically induced preneoplastic lesions in the breast and colon and neoplastic lesions in the skin, forestomach, duodenum and colon of rodents. In addition, curcumin inhibits TPA-induced skin tumor promotion in mice. The mechanisms for the anticarcinogenic effects of curcumin are similar to those of the GTPs. Curcumin enhances glutathione content and glutathione-S-transferase activity in liver; and it inhibits lipid peroxidation and arachidonic acid metabolism in mouse skin, protein kinase C activity in TPA-treated NIH 3T3 cells, chemically induced ODC and tyrosine protein kinase activities in rat colon, and 8-hydroxyguanosine formation in mouse fibroblasts. Ellagic acid is a polyphenol found abundantly in various fruits, nuts and vegetables. Ellagic acid is active in antimutagenesis assays, and has been shown to inhibit chemically induced cancer in the lung, liver, skin and esophagus of rodents, and TPA-induced tumor promotion in mouse skin.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis of ellagic acid O-alkyl derivatives and isolation of ellagic acid as a tetrahexanoyl derivative from Fragaria ananassa.

Ellagic acid [1] is a gallic acid dimer that occurs in plants, fruits, and nuts, either in its free form, or in a series of ellagitannins, or as a glucoside. It has been shown to inhibit cancer induced by several types of chemical carcinogens including polycyclic aromatic hydrocarbons, N-nitrosamines, aflatoxin, and aromatic amines. It has been extracted from a number of fruits, including strawberries; however, its presence in the extracts was determined only by hplc connected with a diode array detector. In the present report, ellagic acid was isolated as a tetrahexanoyl derivative 2 from Fragaria ananassa and identified by 13C and 1H nmr and ms. The 13C-nmr shifts of the aromatic carbons adjacent to a hexanoyloxy group were assigned using two new synthetic model compounds: 3,3'-dihexanoyloxydiphenic-2,2',6,6'-dilactone [3] and 4,4'-dihexanoyloxydiphenic-2,2',6,6'-dilactone [4]. Two new derivatives of ellagic acid [1],3,3'-di-beta-D-glucopyranosylellagic acid decaacetate [5] and 3,3'-di-n-octyl-4,4'-dihexanoylellagic acid [7], were also synthesized. Both derivatives were less effective as inhibitors of benzo[a]pyrene tumorigenesis in the lungs of strain A/J mice than ellagic acid.

The effects of pH and rat intestinal contents on the liberation of ellagic acid from purified and crude ellagitannins.

This study was undertaken to measure the liberation in vitro of ellagic acid [2], a naturally occurring inhibitor of carcinogenesis, from precursor ellagitannins under conditions found in the gut tract. Enzymes, namely beta-glucosidase, esterases, and alpha-amylase, were incubated with raspberry extract. In addition, raspberry extract and casuarictin [1] were treated at different pH's and with the contents of small intestine and cecum from rats fed AIN-76A diet. The esterase activity of the enzyme samples was measured spectrophotometrically using p-nitrophenol acetate as the substrate, and the amount of ellagic acid [2] released from all samples was analyzed by hplc. The hydrolysis of the ellagitannins was not catalyzed by any of the purified enzymes tested, and components of the raspberry extract were found to inhibit the purified esterases noncompetitively. Casuarictin [1] was hydrolyzed to yield high quantities of ellagic acid [2] when placed in buffer at pH 7 and 8, or when incubated with cecal contents for two hours. The release of ellagic acid [2] from the raspberry extract was optimal at pH 8, and maximal release in cecal contents occurred with 1 h. Small intestinal contents had no significant effect on ellagic acid liberation from either casuarictin [1] or raspberry extract.

Comparison of rat liver foci assay and strain A mouse lung tumor assay to detect carcinogens: a review.

A comparison was performed of the results reported in the literature of chemicals tested in the rat liver foci assay and/or in the strain A lung tumor assay to the results of the chemicals tested in long-term carcinogenicity bioassays. The rat liver foci assay was sensitive to 69% of 54 compounds found to be carcinogenic in long-term bioassays and the strain A lung tumor assay to 54% of 93 carcinogens. None of 10 compounds found to be noncarcinogenic in long-term bioassays were active in the rat liver foci assay, while 7 of 23 noncarcinogens (30%) were active in the lung tumor assay. Ten of the 17 carcinogens negative in the rat liver foci assay are believed to exhibit tumor-promoting activity; 3 are direct-acting alkylating agents (dimethylsulfate, epichlorohydrin, and beta-propiolactone); and the remaining 3 are azobenzene, 1,2-dibromoethane, and thioacetamide. Thirty-two of the 43 carcinogens negative in the lung tumor assay were active in either (1) the mouse liver only, (2) the rat and not in the mouse, or (3) in both the rat and mouse liver but not in other organs of the mouse. It is proposed that additional investigations be undertaken to further evaluate the rat liver foci assay and the strain A mouse lung tumor assay as short-term in vivo tests for the demonstration of the carcinogenic potential of genotoxic (mutagenic) chemicals and environmental samples of complex mixtures.

Formation and utilization of methionine by rat liver cells in culture.

The enzyme N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthetase) catalyzes the synthesis of methionine from homocysteine. Methylcobalamin is a cofactor for the reaction. The effects of methionine deprivation and methylcobalamin supplementation on the growth of normal and transformed rat liver epithelial cell lines were determined using growth constants to quantitate cell proliferation. No marked specific requirement by the transformed cell lines for methionine relative to leucine was observed. A sigmoidal relationship, however, was found to exist between growth constants and the logarithms of the amino acid concentrations for both normal and transformed cells. Methylcobalamin stimulated the growth rates of the normal and transformed liver cells in methionine-deficient, homocysteine-containing medium. Growth on methionine was not increased by the addition of methylcobalamin. The growth constants for two normal, two spontaneously transformed, one chemically transformed, and one tumor cell line grown in medium in which methionine was replaced by homocysteine were found to be proportional to the level of methionine synthetase. The results demonstrate the utility of growth quantitation to study the methionine dependency of transformed cells.

Isolation and characterization of epithelial cells from bovine pancreatic duct.

Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 microgram per ml) and insulin (1,5 and 10 microgram per ml) had no effect on the growth of the cells. beta-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 microgram per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro.

Test for carcinogenicity of organic contaminants of United States drinking waters by pulmonary tumor response in strain A mice.

The production of lung adenomas in strain A following multiple i.p. injections of selected organic water contaminants was investigated. Of the 16 contaminants tested, only bromoform produced a pulmonary adenoma response that was significantly greater than the pulmonary adenoma response of vehicle-treated control mice.

Murine pulmonary adenoma bioassay of potentially effective agents against slow-growing solid tumors.

An in vitro-in vivo system for screening potentially effective drugs against solid tumors is described. Drug toxicity to plateau-phase pulmonary adenoma cells is used as an in vitro screen for potential activity against solid tumors, since both plateau phase cultured cell populations and solid tumors are composed predominantly of nondividing cells. The effect of drugs with in vitro activity on the rate of appearance of urethan-induced adenomas on the lung surface of strain A mice in vivo is used to assess drug efficacy in the treatment of solid tumors, taking into consideration drug toxicity to and drug metabolism by the host. Arabinosylcytosine and hydroxyurea were ineffective against plateau phase cells in vitro, even at high concentrations (5 to 10 mg/ml), and did not affect pulmonary adenoma growth in vivo, even at toxic doses (arabinosylcytosine, 80 mg/kg; hydroxyurea, 800 mg/kg), as would be expected with these cell cycle-active drugs. Adriamycin, an effective agent against human solid tumors, was cytotoxic to plateau phase cultured cells (0% survivors at 1 mug/ml), and a dose of 2 mg/kg completely inhibited pulmonary adenoma growth in mice. Thus, this pulmonary adenoma bioassay would appear to effectively select for drugs which may be active against solid tumors in humans.