Health Benefits of Berries: Raspberries

Black raspberries are a rich natural source of chemopreventive phytochemicals. Recent studies have shown that freeze-dried black raspberries inhibit the development of oral, esophageal, and colon cancer in rodents, and extracts of black raspberries inhibit benzo(a)pyrene-induced cell transformation of hamster embryo fibroblasts. However, the molecular mechanisms and the active components responsible for black raspberry chemoprevention are unclear. In this study, we found that 2 major chemopreventive components of black raspberries, ferulic acid and -sitosterol, and a fraction eluted with ethanol (RO-ET) during silica column chromatography of the organic extract of freeze-dried black raspberries inhibit the growth of premalignant and malignant but not normal human oral epithelial cell lines. Another fraction eluted with CH2Cl2/ethanol (DM:ET) and ellagic acid inhibited the growth of normal as well as premalignant and malignant human oral cell lines. We investigated the molecular mechanisms by which ferulic acid and -sitosterol and the RO-ET fraction selectively inhibited the growth of premalignant and malignant oral cells using flow cytometry and Western blotting of cell cycle regulatory proteins. There was no discernable change in the cell cycle distribution following treatment of cells with the RO-ET fraction. Premalignant and malignant cells redistributed to the G₂/M phase of the cell cycle following incubation with ferulic acid. -sitosterol treated premalignant and malignant cells accumulated in the G₀/G₁ and G₂/M phases, respectively. The RO-ET fraction reduced the levels of cyclin A and cell division cycle gene 2 (cdc2) in premalignant cells and cyclin B1, cyclin D1, and cdc2 in the malignant cell lines. This fraction also elevated the levels of p21^waf1/cip1 in the malignant cell line. Ferulic acid treatment led to increased levels of cyclin B1 and cdc2 in both cell lines, and p21^waf1/cip1 was induced in the malignant cell line. -sitosterol reduced the levels of cyclin B1 and cdc2 while increasing p21^waf1/cip1 in both the premalignant and malignant cell lines. These results show for the first time that the growth inhibitory effects of black raspberries on premalignant and malignant human oral cells may reside in specific components that target aberrant signaling pathways regulating cell cycle progression.

Oral cavity cancers represent 2.5% of the cancers that occur in the United States and are ranked sixth worldwide. Since current therapeutic protocols are relatively ineffective, alternative strategies for prevention need to be developed and tested in appropriate animal models. In the study reported herein, the hamster cheek pouch (HCP) was used to evaluate the ability of black raspberries to inhibit oral cavity tumors. Male Syrian Golden hamsters, 3-4 weeks of age, were fed 5% and 10% lyophilized black raspberries (LBR) in the diet for two weeks prior to treatment with 0.2% 7,12-dimethylbenz(a) anthracene in dimethylsulfoxide and for 10 weeks thereafter. HCPs were painted 3X/week for eight weeks. The animals were sacrificed 12-13 weeks from the beginning of DMBA treatment and the number and volume of tumors (mm3) determined. There was a significant difference (p = 0.02) in the number of tumors between the 5% LBR and control groups (27 tumors/14 animals and 48 tumors/15 animals, respectively) and an intermediate number of tumors in the 10% berry-treated animals (39 tumors/15 animals). These experiments support previous studies from our laboratories showing the chemopreventive activity of black raspberries and show, for the first time, that dietary black raspberries will inhibit tumor formation in the oral cavity.

Freeze-dried black raspberries have been shown to inhibit the development of chemically induced esophageal and colon cancer in rodents.In addition, organic extracts of black raspberries inhibit benzo(a)pyrene (BaP)-induced cell transformation in vitro. The molecular mechanisms through which black raspberries inhibit carcinogenesis remain unclear. We investigated the effects of black raspberry extracts on transactivation of activated protein 1 (AP-1) and nuclear factor B (NFB) induced by BaP diol-epoxide (BPDE), the ultimate carcinogen of BaP, in mouse epidermal JB6 Cl 41 (Cl 41) cells. Black raspberries were extracted with methanol, and the methanol extract was partitioned and chromatographed into several fractions designated RU-F003, RU-F004, RU-DM, and RU-ME. Pretreatment of Cl 41 cells with RU-F003, RU-DM, or RU-ME resulted in an inhibition of BPDE-induced AP-1 and NFB activities. The RU-ME fraction was the most potent inhibitor among the fractions tested. In contrast, fraction RU-F004 did not inhibit BPDE-induced AP-1 or NFB activities in Cl 41 cells. The inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NFB appear to be mediated via inhibition of mitogen activated protein kinase activation and inhibitory subunit B phosphorylation, respectively. Pretreatment of cells with berry fractions did not result in an inhibition of BPDE binding to DNA; thus, this was not a mechanism of reduced AP-1 and NFB activities. None of the fractions was found to affect p53-dependent transcription activity. In view of the important roles of AP-1 and NFB in tumor promotion/progression, these results suggest that the ability of black raspberries to inhibit tumor development may be mediated by impairing signal transduction pathways leading to activation of AP-1 and NFB. The RU-ME fraction appears to be the major fraction responsible for the inhibitory activity of black raspberries.

This study examined the effects of lyophilized black raspberries (BRB) on azoxymethane (AOM)-induced aberrant crypt foci (ACF), colon tumors, and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in male Fischer 344 rats. AOM was injected (15 mg/kg body wt ip) once per week for 2 wk. At 24 h after the final injection, AOM-treated rats began consuming diets containing 0%, 2.5%, 5%, or 10% (wt/wt) BRB. Vehicle controls received 5% BRB or diet only. Rats were sacrificed after 9 and 33 wk of BRB feeding for ACF enumeration and tumor analysis. ACF multiplicity decreased 36%, 24%, and 21% (P < 0.01 for all groups) in the 2.5%, 5%, and 10% BRB groups, respectively, relative to the AOM-only group. Total tumor multiplicity declined 42%, 45%, and 71% (P < 0.05 for all groups). Although not significant, a decrease in tumor burden (28%, 42%, and 75%) was observed in all BRB groups. Adenocarcinoma multiplicity decreased 28%, 35%, and 80% (P < 0.01) in the same treatment groups. Urinary 8-OHdG levels were reduced by 73%, 81%, and 83% (P < 0.01 for all groups). These results indicate that BRB inhibit several measures of AOM-induced colon carcinogenesis and modulate an important marker of oxidative stress in the Fischer 344 rat.

Fruit and vegetable consumption has consistently been associated with decreased risk of a number of aerodigestive tract cancers, including esophageal cancer. We have taken a "food-based" chemopreventive approach to evaluate the inhibitory potential of lyophilized black raspberries (LBRs) against N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in the F344 rat, during initiation and postinitiation phases of carcinogenesis. Anti-initiation studies included a 30-week tumorigenicity bioassay, quantification of DNA adducts, and NMBA metabolism study. Feeding 5 and 10% LBRs, for 2 weeks prior to NMBA treatment (0.25 mg/kg, weekly for 15 weeks) and throughout a 30-week bioassay, significantly reduced tumor multiplicity (39 and 49%, respectively). In a short-term bioassay, 5 and 10% LBRs inhibited formation of the promutagenic adduct O⁶-methylguanine (O⁶-meGua) by 73 and 80%, respectively, after a single dose of NMBA at 0.25 mg/kg. Feeding 5% LBRs also significantly inhibited adduct formation (64%) after NMBA administration at 0.50 mg/kg. The postinitiation inhibitory potential of berries was evaluated in a second bioassay with sacrifices at 15, 25, and 35 weeks. Administration of LBRs began after NMBA treatment (0.25 mg/kg, three times per week for 5 weeks). LBRs inhibited tumor progression as evidenced by significant reductions in the formation of preneoplastic esophageal lesions, decreased tumor incidence and multiplicity, and reduced cellular proliferation. At 25 weeks, both 5 and 10% LBRs significantly reduced tumor incidence (54 and 46%, respectively), tumor multiplicity (62 and 43%, respectively), proliferation rates, and preneoplastic lesion development. Yet, at 35 weeks, only 5% LBRs significantly reduced tumor incidence and multiplicity, proliferation indices and preneoplastic lesion formation. In conclusion, dietary administration of LBRs inhibited events associated with both the initiation and promotion/progression stages of carcinogenesis, which is promising considering the limited number of chemopreventives with this potential.

Freeze-dried fruits of two strawberry cultivars, Sweet Charlie and Carlsbad, and two blueberry cultivars, Tifblue and Premier, were sequentially extracted with hexane, 50% hexane/ethyl acetate, ethyl acetate, ethanol, and 80% acetone/water at ambient temperature. Solvents were then evaporated at 40° C. The extracts were tested against in vitro cultures of two aggressive cervical cancer cell lines (CaSki and SiHa) and two breast cancer cell lines with different requirements for estrogen (MCF-7 and T47-D). The metabolic activity of the cultures was assessed with MTS viability assay. The Ames assay for mutagenesis was used to evaluate the anticarcinogenic effects of phytochemicals with a 90% accuracy. Ethanol extracts from all four fruits strongly inhibited CaSki and SiHa cervical cancer cell lines and MCFF-7 and T47-D breast cancer cell lines. An unfractionated aqueous extract of raspberry and the ethanol extract of Premier blueberry significantly inhibited mutagenesis by both direct-acting and metabolically activated carcinogenesis.

This study examined the effects of lyophilized black raspberries (BRB) on azoxymethane (AOM)-induced aberrant crypt foci (ACF), colon tumors, and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in male Fischer 344 rats. AOM was injected (15 mg/kg body wt ip) once per week for 2 wk. At 24 h after the final injection, AOM-treated rats began consuming diets containing 0%, 2.5%, 5%, or 10% (wt/wt) BRB. Vehicle controls received 5% BRB or diet only. Rats were sacrificed after 9 and 33 wk of BRB feeding for ACF enumeration and tumor analysis. ACF multiplicity decreased 36%, 24%, and 21% (P < 0.01 for all groups) in the 2.5%, 5%, and 10% BRB groups, respectively, relative to the AOM-only group. Total tumor multiplicity declined 42%, 45%, and 71% (P < 0.05 for all groups). Although not significant, a decrease in tumor burden (28%, 42%, and 75%) was observed in all BRB groups. Adenocarcinoma multiplicity decreased 28%, 35%, and 80% (P < 0.01) in the same treatment groups. Urinary 8-OHdG levels were reduced by 73%, 81%, and 83% (P < 0.01 for all groups). These results indicate that BRB inhibit several measures of AOM-induced colon carcinogenesis and modulate an important marker of oxidative stress in the Fischer 344 rat