The present study used a postinitiation protocol to investigate molecular mechanisms by which black raspberries (BRBs) influence the late stages of N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in rats. F344 rats were injected with NMBA and then fed either control diet or a diet containing 5% BRB powder. Control rats were injected with DMSO/water (20:80), the vehicle for NMBA. Esophagi from control, NMBA- and NMBA_BRB-treated rats were collected at 35 wk for histopathological, molecular, and immunohistochemical analyses. Treatment with 5% BRBs reduced the number of dysplastic lesions and the number and size of esophageal papillomas in NMBA-treated rats. When compared to esophagi from control rats, NMBA treatment led to the differential expression of 4807 genes in preneoplastic esophagus (PE) and 17 846 genes in esophageal papillomas. Dietary BRBs modulated 626 of the 4807 differentially expressed genes in PE and 625 of the 17 846 differentially expressed genes in esophageal papillomas towards normal levels of expression. In both PE and in papillomas, BRBs modulated the mRNA expression of genes associated with carbohydrate and lipid metabolism, cell proliferation and death, and inflammation. In these same tissues, BRBs modulated the expression of proteins associated with proliferation, apoptosis, inflammation, angiogenesis, and both cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism. Interestingly, matrix metalloproteinases involved in tissue invasion and metastasis, and proteins associated with cell-cell adhesion, were also modulated by BRBs. This is the first report of the effects of berries on the expression of genes associated with the late stages of rat esophageal carcinogenesis. (C) 2011 Wiley-Liss, Inc.

This study was undertaken to determine if the oral consumption of red beetroot food color would result in an inhibition of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. Rats were treated with NMBA and given either regular water ad libitum or water containing 78 microg/mL commercial red beetroot dye, E162. The number of NMBA-induced esophageal papillomas was reduced by 45% (P < .001) in animals that received the food color compared to controls. The treatment also resulted in reduced rates of cell proliferation in both precancerous esophageal lesions and in papillomas of NMBA-treated rats, as measured by immunohistochemical staining of Ki-67 in esophageal tissue specimens. The effects of beetroot food color on angiogenesis (microvessel density by CD34 immunostaining), inflammation (by CD45 immunostaining), and apoptosis (by terminal deoxynucleotidyl transferase dUTP nick end-labeling staining) in esophageal tissue specimens were also determined. Compared to rats treated with NMBA only, the levels of angiogenesis and inflammation in the beetroot color-consuming animals were reduced, and the apoptotic rate was increased. Thus, the mechanism(s) of chemoprevention by the active constituents of red beetroot color include reducing cell proliferation, angiogenesis, and inflammation and stimulating apoptosis. Importantly, consumption of the dye in the drinking water for a period of 35 weeks did not appear to induce any overt toxicity. Based on the fact that red beetroot color contains betanins, which have strong antioxidant activity, it is postulated that these effects are mediated through inhibition of oxygen radical-induced signal transduction. However, the sum of constituents of E162 has not been determined, and other components with other mechanisms may also be involved in antagonizing cancer development.

Abstracts: Frontiers in Cancer Prevention Research 2008 A131 We reported that the feeding of a diet containing 5 or 10% freeze-dried black raspberries (BRB) to N -nitrosomethylbenzylamine (NMBA)-treated rats results in a 39-64% reduction in the number of esophageal papillomas when using either anti-initiation or anti-promotion/progression protocols. The molecular events associated with the effects of BRB on NMBA-induced preneoplastic and papillomatous esophageal lesions however, have not been fully elucidated. In the present study, 4-5 week-old male F344 rats were injected s.c. with NMBA (0.3 mg/kg b.w., 3x/wk for 5 wks) after which they were fed either control diet or diet containing 5% BRB until the end of the study (35 wks). Control rats were injected s.c. with a solution of DMSO/water (20:80), the vehicle for NMBA. Esophagi from vehicle control, NMBA- and NMBA + BRB-treated rats were collected at 35 weeks for histologic grading, and for microarray and Real-Time PCR analyses. Treatment with 5% BRB reduced the number of preneoplastic lesions (dysplasias) and the number and size of papillomas in the esophagus of NMBA-treated rats. When compared to esophagi from vehicle control rats, NMBA treatment alone led to the differential expression of 4,807 genes in preneoplastic esophagus and 17,846 genes in esophageal papillomas. Treatment with 5% BRB resulted in a modulation towards control levels of expression of 626 genes in preneoplastic esophagus and 627 genes in papillomas. In both preneoplastic esophagus and in papillomas, the berry-modulated genes were associated with regulation of cell proliferation, inflammation and receptor-mediated pathways. Twenty-five genes were commonly modulated (down- or up-regulated) by BRB in both preneoplastic lesions and in papillomas. Interestingly, several of these genes are associated with matrix metalloproteinases involved in tissue invasion and metastasis, cell-cell adhesion and with calcium signaling. This is the first report suggesting that berries might influence genes involved in tissue invasion and metastasis in the rat esophagus. (Supported by NCI grant No. CA103180) Citation Information: Cancer Prev Res 2008;1(7 Suppl):A131.

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-kappaB 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 approximately 3.8 micromol anthocyanins/g diet), (d) an organic-insoluble (residue) fraction (containing 0.02 mumol 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.