Cancer Prevention Research Anthocyanins in Black Raspberries Prevent Esophageal Tumors in Rats

Diets containing freeze-dried black raspberries (BRB) suppress the development of N-nitrosomethylbenzylamine (NMBA)–induced tumors in the rat esophagus. Using bioassay-directed fractionation, the anthocyanins in BRB were found to be the most active constituents for down-regulation of carcinogen-induced nuclear factor-κB and activator protein-1 expression in mouse epidermal cells in vitro. The present study was undertaken, therefore, to determine if the anthocyanins contribute to the chemopreventive activity of BRB in vivo. F344 rats consumed diets containing either (a) 5% whole BRB powder, (b) an anthocyanin-rich fraction, (c) an organic solvent-soluble extract (a–c each contained ∼3.8 μmol anthocyanins/g diet), (d) an organic-insoluble (residue) fraction (containing 0.02 μmol anthocyanins/g diet), (e) a hexane extract, and (f) a sugar fraction (e and f had only trace quantities of anthocyanins), all derived from BRB. Animals were fed diets 2 weeks before treatment with NMBA and throughout the bioassay. Control rats were treated with NMBA only. Animals were killed at week 30, and esophageal tumors were enumerated. The anthocyanin treatments (diet groups a–c) were about equally effective in reducing NMBA tumorigenesis in the esophagus, indicating that the anthocyanins in BRB have chemopreventive potential. The organic-insoluble (residue) fraction (d) was also effective, suggesting that components other than berry anthocyanins may be chemopreventive. The hexane and sugar diets were inactive. Diet groups a, b, and d all inhibited cell proliferation, inflammation, and angiogenesis and induced apoptosis in both preneoplastic and papillomatous esophageal tissues, suggesting similar mechanisms of action by the different berry components. Esophageal cancer is the third most common gastrointestinal malignancy (1) and the sixth most frequent cause of cancer death in the world (2). Squamous cell carcinoma is the predominant histologic subtype worldwide, and persons with this disease have a high rate of mortality (3). It has been estimated that more than two thirds of human cancer can be prevented through appropriate lifestyle modifications (4). Although Doll and Peto (5) reported that ∼35% of human cancer mortality is caused by diet, more than 250 population-based studies, including case-control and cohort studies, indicate that persons who eat about five servings of fruit and vegetables per day have approximately half the risk of developing cancer—particularly cancers of the digestive and respiratory tracts—than do those who eat fewer than two servings per day (4). Chemoprevention can play an integral role in the overall strategy of reducing the incidence of cancer and is a potentially viable approach for reducing the risk of esophageal cancer in high-risk individuals (6). Experimental studies provide evidence that dietary compounds prevent cancer through multiple mechanisms (4). These include the induction of cellular detoxifying and antioxidant enzymes, which protect against cellular damage caused by carcinogens and endogenously generated reactive oxygen species. Dietary compounds can also reduce cell proliferation rates, inflammation, and angiogenesis and stimulate apoptosis, normal cell differentiation, and cell-cell adhesion, among other effects (4). Because the process of cancer development involves multiple stages (i.e., initiation and promotion/progression), chemopreventive compounds have been classified broadly as blocking agents, which impede the initiation stage, or as suppressing agents, which arrest or reverse the promotion/progression stage (7). Anthocyanins are naturally occurring polyphenolic compounds that provide pigmentation to many fruits and vegetables, such as berries, red grapes, purple sweet potato, and red cabbages (8). Berries are rich in anthocyanins, which account for ∼5% to 10% of their dry weight (9). We and others have suggested that anthocyanins may be cancer preventive because they (a) inhibit cell transformation, in part, by blocking Authors' Affiliations: The Ohio State University Comprehensive Cancer Center and Department of Physical Activity and Educational Services, The Ohio State University, Columbus, Ohio; and Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota Received 08/05/2008; revised 09/26/2008; accepted 11/05/2008. Grant support: National Cancer Institute grant CA103180 and U.S. Department of Agriculture grant 38903-03560. Requests for reprints: Gary D. Stoner, Department of Internal Medicine, Ohio State University Comprehensive Cancer Center, 2001 Polaris Parkway, Columbus, OH 43240. Phone: 614-293-3268; Fax: 614-293-5952; E-mail: [email protected]. ©2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0155 84 Cancer Prev Res 2009;2(1) January 2009 www.aacrjournals.org Cancer Research. on April 9, 2017. © 2009 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from the mitogen-activated protein kinase pathway and activator protein-1 expression (10); (b) suppress inflammation by blocking the nuclear factor-κB (NF-κB) pathway and cyclooxygenase 2 (COX-2) gene expression (10); and (c) induce apoptosis in cancer cells through the activation of reactive oxygen species/ c-Jun NH2-terminal kinase–mediated caspases (8). The most commonly used preclinical animal model for esophageal squamous cell carcinoma is the F344 rat in which esophageal papillomas are induced by the nitrosamine carcinogen, N-nitrosomethylbenzylamine (NMBA; ref. 11). NMBA is administered by s.c. injection (0.25-0.5 mg/kg body weight/ injection) either thrice per week for 5 weeks or once per week for 15 weeks; this results in a 100% incidence of esophageal papillomas at 25 weeks (12). In separate studies, our laboratory found that the feeding of a diet containing 5% or 10% BRB to NMBA-treated rats produced a 39% to 64% reduction in the number of esophageal papillomas when using either anti-initiation or antipromotion/progression protocols (13). We also reported that an organic solvent–soluble extract of BRB down-regulates genes associated with proliferation, inflammation, and angiogenesis in cultured mouse epidermal JB-6 Cl 41 cells (10). In a study in which 168 subfractions of this extract were tested individually for their ability to down-regulate anti–benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)–induced NF-κB activity in JB-6 cells, the three most abundant anthocyanins [cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-(2-xylosylrutinoside)] in BRB were found to have the highest inhibitory activity (14). The role of these anthocyanins in the prevention of esophageal tumors in vivo, however, has not been determined. The present study was conducted, therefore, to determine whether the anthocyanins in BRB play a role in the ability of the berries to prevent NMBA-induced tumors in the rat esophagus. We also determined if diets that vary in anthocyanin contents produce similar or different effects on cell proliferation, apoptosis, inflammation, and angiogenesis in NMBA-treated esophageal tissues having varying extents of neoplastic change. Materials and Methods Source of black raspberries and freeze drying Black raspberries (Rubus occidentalis; BRB) of the Jewel variety were obtained from a single farm in Ohio. Ripe berries were picked mechanically, washed, and frozen at −20°C within 2 h of the time of picking. The berries were then taken to Van Drunen Farms for freezedrying and grinding into a powder as described (13). The berry powder was then shipped frozen to the Ohio State University where it was stored at −20°C. A 100-g sample of the berry powder was shipped to Covance Laboratories for analysis of certain vitamins, minerals, carotenoids, phenolic acids, phytosterols, fungicides, pesticides, and herbicides as described (13). BRB powder was also shipped frozen to the laboratory of Dr. Stephen Hecht for preparation of BRB extracts and fractions as described below. The remaining BRB powder was stored frozen and used in an esophageal carcinogenesis bioassay conducted at the Ohio State University (also described below). Preparation of extracts from BRB A schematic illustrating the steps used for preparation of extracts from BRB is shown in Fig. 1. Hexane extract. BRB (500 g) were placed in a 2,500 mL beaker with 1,500 mL hexane, and the mixture was stirred with an explosion proof mechanical stirrer and sonicated for 30 min at room temperature. The supernatant was vacuum-filtered through a Buchner funnel. This procedure was repeated twice using fresh 1,500 mL volumes of hexane, Fig. 1. Fractionation scheme for the preparation of extracts from freeze-dried BRB. Anthocyanins Are Chemopreventive for Rat Esophagus 85 Cancer Prev Res 2009;2(1) January 2009 www.aacrjournals.org Cancer Research. on April 9, 2017. © 2009 American Association for cancerpreventionresearch.aacrjournals.org Downloaded from for a total extract volume of 4,500 mL. After the final extraction, the residue was dried briefly on the funnel with a stream of N2. The combined hexane extracts were concentrated to dryness by rotary evaporation at reduced pressure. The final extract was stored at −20°C. Ethanol/H2O:80/20–soluble extract. The residue from the hexane extract was placed in a 2,500 mL beaker with 1,500 mL of ethanol/ H2O:80/20, and the mixture was stirred and sonicated as above for 30 min at room temperature. The resulting mixture was filtered as above, and the procedure was repeated three more times with fresh 1,500 mL batches of ethanol/H2O:80/20. The solvents were removed to the extent possible by rotary evaporation at reduced pressure. This yielded a syrup. The remaining solvent was removed by freeze drying, as described below. Ethanol/H2O–insoluble (residue) fraction. The berry mass obtained above after removal of the ethanol/H2O:80/20 solvent was allowed to dry for several minutes under vacuum, placed in a vacuum dessicator for 4 d, and then stored at 4°C. This fraction represents the solids in BRB and, from this point on, will be called the “residue” fraction. Anthocyanin fraction and sugar fraction A polystyrene-based resin, SP-700 (Mitsubishi Chemical Co.; 1.5 liters, 825 g) was activated by overnight treatment with 1.2 liters of 96% ethanol, followed by rinsing with 1.2 liters of doubly distilled H2O. The resin was stirred in the H2O for 10 min and then filtered. The filtered resin was mixed with 1.5 liters of doubly distilled H2O and introduced into a 7.5 cm (diameter) × 50 cm glass column containing a glass frit. The height of the resin in the column was about 36 cm. The packed column was washed thoroughly with 4 liters of H2O to remove any ethanol. The washed resin was poured back into a 3-liter beaker, and about 150 g of the ethanol/H2O:80/20 extract (as a concentrated syrup equivalent to about 93 g of the dried extract, or 11% of the resin weight) was then added. The mixture was stirred for 10 min to allow the anthocyanins to adsorb on the resin and then put back into the column. The column was eluted with about 2 liters of H2O at a rate of about 20 mL/min. This eluant contained the “sugar fraction.” Then, 3 liters of 95% ethanol was added to the column in steps. The first 800 mL, containing H2O that was already on the column, was collected separately and combined with the sugar fraction. The next 2 liters of the now deeply colored ethanol eluant was collected; it contained the anthocyanin fraction. The anthocyanin fraction was concentrated to a tar-like consistency using a centrifugal vacuum concentrator (Speed Vac, Thermo Savant) using a preparative rotor. The sugar fraction was concentrated by rotary evaporation under reduced pressure. Freeze drying of the ethanol/H20:80/20–soluble extract and sugar fractions. During conventional freeze drying, the sugar fraction tended to remain as a syrup and the 80/20 extract expanded to a semisolid tar. Special techniques were necessary, therefore, to obtain these as dry fractions. The initial water content of each of these fractions was determined by freeze drying or rotary evaporating a small aliquot to dryness. The fractions were then adjusted to approximately 75% to 80% H2O content. A metal colander with numerous ∼4-mm circular openings in the bottom was placed over a stainless-steel beaker, which was approximately two thirds full with 4 liters of liquid N2. Seven hundred milliliters of the sugar or the ethanol/H2O:80/20 fraction were poured into the colander over a 2-min period. The majority of the extract formed 5-mm frozen spheres at the bottom of the beaker. The liquid N2 and the spheres were poured through a wire mesh strainer. The spheres in the strainer were immediately placed in a precooled (−80°C) 10 × 6 × 1.2-in. metal tray. The tray was placed on a prechilled (−25°C) shelf in a freeze drier and vacuum was applied. The sample remained under these conditions for 4 d. The freezing shelf temperature was then allowed to equilibrate, and the sample remained under these conditions for 3 d. The result was a dry foam for the ethanol/H2O:80/20 extract and the sugar fraction. These fractions were then stored at −20°C. Anthocyanin levels in BRB, the ethanol/H2O:80/20 extract, and the anthocyanin fraction were determined by high-performance liquid chromatography before incorporation in the diet (14). The molar percents of the three major anthocyanins in BRB were 12% cyanidin-3-O-glucoside, 34% cyanidin-3-O-(2-xylosylrutinoside), and 53% cyanidin-3-O-rutinoside. These molar percents were very similar for the ethanol/water-soluble-soluble extract and the anthocyanin fraction.