Suppression of Oxidative Stress and NFκB/MAPK Signaling by Lyophilized Black Raspberries for Esophageal Cancer Prevention in Rats

Abstract

Research in the laboratory has shown that lyophilized black raspberries (BRB) significantly inhibit N-nitrosomethylbenzylamine (NMBA)-induced esophageal squamous cell carcinogenesis in rats. The objective of the present study is to characterize the underlying mechanism(s) of anti-cancer action of BRB in this preclinical animal model focusing on oxidative stress and its related oncogenic signaling pathways. Esophageal epithelial tissues were collected and assessed for markers of oxidative stress and nuclear factor κB (NFκB) and mitogen-activated protein kinase (MAPK). BRB reduced the incidence of esophageal cancer from 100% in NMBA-treated rats to 81.5% in rats treated with NMBA plus BRB (p < 0.05). Tumor multiplicity was reduced from 4.73 ± 0.45 tumors per esophagus in NMBA-treated rats to 1.44 ± 0.26 in rats treated with NMBA plus BRB (p < 0.001). The data indicated that NMBA treatment increased production of hydrogen peroxide and lipid hydroperoxide, reduced expression and activity of glutathione peroxidase and superoxide dismutase 2, and activated NFκB/MAPK signaling in rat esophagus. The study's results show that BRB reverses oxidative stress and suppresses NFκB/MAPK pathways, which could be the mechanisms for esophageal cancer chemopreventive action of BRB in rats.

Keywords: MAPK; NFκB; black raspberries; esophageal cancer; oxidative stress.

Figure 1

Black raspberries (BRB) decreases level of 8-OxoG. (A) Immunohistochemistry (IHC) staining of 8-OxoG in normal animals and rats treated with N-nitrosomethylbenzylamine (NMBA) or NMBA + BRB (200×). The upper right image is the version under 100×; and (B) statistical analysis of 8-OxoG staining. The values are expressed as mean; bars, ±SE. * p < 0.05.

Figure 2

BRB increases expression levels of glutathione peroxidase (GPx) and superoxide dismutase 2 (SOD2). (A,B), mRNA expression of GPx and SOD2 in rat esophagus; (C,D), protein expression of GPx and SOD2 in rat esophagus. The values are expressed as mean; bars, ±SE. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

BRB suppressed activation of nuclear factor κB (NFκB) and mitogen-activated protein kinase (MAPK) signaling. (A) Western blot detected phosphorylation of extracellular signal–regulated kinases (ERK), p38 MAPK, stress-activated protein kinase/Jun amino-terminal kinases (SAPK/JNK), NFκB-p65, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), and IκB kinase alpha/beta (IKK)α/β in rat esophagus; and (B) quantitative analysis of western blot. The values are expressed as mean; bars, ±SE. **** p < 0.0001.

Figure 4

Correlations between H₂O₂ production and phosphorylation of NFκB p65 (A) or p38 MAPK (B) in NMBA-treated rat esophagus.

Figure 5

Possible mechanisms of action of BRB in esophageal cancer prevention. Upper figure, BRB modulates oxidative stress/antioxidant pathway; lower figure, BRB inhibits MAPK and NFκB activation. Arrows indicate activation and T-lines indicate inhibition. L-Arg: L-arginine; iNOS: inducible nitric oxide synthase; ONOO: Peroxynitrite; GSSG: glutathione disulfide; NADPH: nicotinamide adenine dinucleotide phosphate reduced form; GSH: hydroperoxides using glutathione; GR: glutathione reductase; ROS/RNS: reactive oxygen species and reactive nitrogen species; P: phosphorylation; MEK: mitogen-activated protein kinase kinase; MKK: mitogen-activated protein kinase kinase; Erk: extracellular-signal-regulated kinases; COX-2: cyclooxygenase 2; PGE₂: prostaglandin E2.