Chemopreventive properties of black raspberries in N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis: down-regulation of cyclooxygenase-2, inducible nitric oxide synthase, and c-Jun

Abstract

Our laboratory has used a rodent model of human esophageal squamous cell carcinoma to identify putative chemopreventive agents for this disease and to determine their mechanisms of action. In the present study, we treated F344 rats with the esophageal carcinogen, N-nitrosomethylbenzylamine (NMBA), thrice per week for 5 weeks. Beginning 1 week later, they were fed a synthetic diet containing 5% black raspberries (BRB) for the duration of the bioassay (25 weeks). Rats were sacrificed at weeks 9, 15, and 25. Esophageal tissues were collected, and tumor data were recorded. The expression and enzymatic activities of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of c-Jun in the esophagi, were evaluated to investigate the mechanism(s) by which black raspberries modulate tumorigenesis. At week 25, BRB inhibited tumor multiplicity, the standard end point in this tumor model, from 3.78 +/- 0.41 tumors per rat in NMBA-treated animals to 2.23 +/- 0.21 tumors per rat in animals treated with NMBA plus BRB (P < 0.005). BRB reduced mRNA and protein expression levels of COX-2, iNOS, and c-Jun as well as the level of prostaglandin E(2) in preneoplastic lesions of the esophagus at week 25. The berries inhibited mRNA expression of iNOS and c-Jun, but not COX-2, in papillomatous lesions of the esophagus. Prostaglandin E(2) and total nitrite levels were also decreased by BRB in papillomas. These results suggest a novel tumor suppressive role of BRB through inhibition of COX-2, iNOS, and c-Jun.

Figures

Figure 1

Effect of BRB on tumor development in NMBA-treated rat esophagus at 25 weeks. Rats treated with NMBA + BRB had fewer tumors than rats treated with NMBA only (A); however, there was no significant difference in tumor volume between the two groups of rats (B). ●, NMBA control; △, NMBA+BRB.

Figure 2

Fold changes for iNOS (A), COX-2 (B), and c-Jun (C) mRNA expression in rat esophageal preneoplastic lesions at weeks 9, 15, and 25; and in papillomas at week 25 (D). Columns, means; bars, ± SE; *, P < 0.05 as determined by ANOVA when compared with the control diet group. □, Normal control; , NMBA control; , NMBA+BRB.

Figure 3

Effect of BRB on the protein expression of iNOS, COX-2, and c-Jun in preneoplastic lesions at week 25. The indicated proteins were detected by Western blot analyses. Representative blots are shown, similar results were obtained from triplicate experiments. Columns, mean relative densitometric intensities; bars, ± SE; *, P < 0.05; **, P < 0.005 as determined by ANOVA when compared with the control diet group. □, Normal control; , NMBA control; , NMBA+BRB.

Figure 4

Possible mechanisms for the prevention of tumor development in rat esophagus by BRB.