Differential effects of black raspberry and strawberry extracts on BaPDE-induced activation of transcription factors and their target genes

Abstract

The chemopreventive properties of edible berries have been demonstrated both in vitro and in vivo, however, the specific molecular mechanisms underlying their anti-cancer effects are largely unknown. Our previous studies have shown that a methanol extract fraction of freeze-dried black raspberries inhibits benzoapyrene (BaP)-induced transformation of Syrian hamster embryo cells. This fraction also blocks activation of activator protein-1 (AP-1) and nuclear factor kappaB (NF-kappaB) induced by benzoapyrene diol-epoxide (BaPDE) in mouse epidermal JB6 Cl 41 cells. To determine if different berry types exhibit specific mechanisms for their anti-cancer effects, we compared the effects of extract fractions from both black raspberries and strawberries on BaPDE-induced activation of various signaling pathways in Cl 41 cells. Black raspberry fractions inhibited the activation of AP-1, NF-kappaB, and nuclear factor of activated T cells (NFAT) by BaPDE as well as their upstream PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. In contrast, strawberry fractions inhibited NFAT activation, but did not inhibit the activation of AP-1, NF-kappaB or the PI-3K/Akt-p70(S6K) and mitogen-activated protein kinase pathways. Consistent with the effects on NFAT activation, tumor necrosis factor-alpha (TNF-alpha) induction by BaPDE was blocked by extract fractions of both black raspberries and strawberries, whereas vascular endothelial growth factor (VEGF) expression, which depends on AP-1 activation, was suppressed by black raspberry fractions but not strawberry fractions. These results suggest that black raspberry and strawberry components may target different signaling pathways in exerting their anti-carcinogenic effects.

Figure 1

Inhibition of BaPDE-induced activation of AP-1 and NFκB by fractions from black raspberries but not strawberries. Mouse epidermal Cl 41 cells stably transfected with AP-1 luciferase (A and B) or with NFκB luciferase (C and D) were seeded into each well of 96-well plates at 8 × 10³ cells/well and cultured in 5% FBS MEM at 37°C for 12 h. The cells were pretreated with various fractions of black raspberry extracts (A and C) or strawberry extracts (B and D) for 30 min and then exposed to BaPDE (2 μM) for AP-1 and NFκB induction for 12 h. The cells were extracted with lysis buffer, and luciferase activity was measured using Promega luciferase assay reagent with a luminometer after the addition of 50 μl of lysis buffer for 30 min at 4°C. Results are presented as AP-1- or NFκB-dependent transcriptional activity relative to medium control (relative AP-1 or NFκB activity). Each bar indicates the mean and standard error of four repeat assay wells. The asterisk (*) indicates a significant decrease from BaPDE treatment alone (P <0.05). DM and ME refer to non-polar and polar fractions of a silica gel column fractionation.

Figure 2

Effects of black raspberry and strawberry fractions on VEGF induction by BaPDE. Mouse epidermal Cl 41 cells stably transfected with VEGF luciferase were seeded into each well of 96-well plates at 8 × 10³ cells/well and cultured in 5% FBS MEM at 37°C for 12 h. The cells were pretreated with various fractions of strawberry extracts (A and C) or black raspberry extracts (B and C) as indicated for 30 min and then exposed to BaPDE (2 μM) for VEGF induction. After 24 h incubation, cells were extracted with lysis buffer, and luciferase activity was measured. Results are presented as VEGF induction relative to medium control (relative VEGF induction). Each bar indicates the mean and standard error of four repeat assay wells. The asterisk (*) indicates a significant decrease from BaPDE treatment alone (P <0.05).

Figure 3

Comparison of effects of RO-ME and FA-ME on BaPDE-induced activation of PI-3K, Akt, p70^S6K, and MAPKs. Cl 41 cells were seeded into 100-mm tissue culture dishes (A) or each well of 6-well plates (B and C), and cultured in 5% FBS MEM at 37°C until cell density reached 70–80%. The cell culture medium was replaced with 0.1% FBS MEM. Forty-five hours later, cells were incubated with fresh serum-free MEM for 3–4 h at 37°C. Cells (A) were then pretreated with the indicated berry fractions at 50 μg/ml for 30 min, exposed to BaPDE (2 μM) for 30 min, and lysed in 400 μl lysis buffer. PI-3K activity was measured as described in Materials and Methods; or (B and C) were pretreated with indicated berry fractions at 12 μg/ml for 30 min, exposed to BaPDE (2 μM) for 270 min, and extracted with an SDS-sample buffer and Western blot analysis carried out as described in Materials and Methods.

Figure 4

Effects of berry fractions on BaPDE-induced NFAT transactivation and TNF-α expression. Mouse epidermal Cl 41 cells stably transfected with NFAT luciferase or TNF-α luciferase were seeded into each well of 96-well plates at 8 × 10³ cells/well and cultured in 5% FBS MEM at 37°C for 12 h. The cells were pretreated with various fractions of strawberry extracts (A, C, and D) or black raspberry extracts (B and D) as indicated for 30 min at the concentrations indicated, and then exposed to BaPDE (2 μM) for and luciferase activity was measured using Promega luciferase assay reagent with a luminometer. Results are presented as NFAT activity or TNF-α induction relative to medium control (relative NFAT activity or relative TNF-α induction). Each bar indicates the mean and standard error of four repeat assay wells. The asterisk (*) indicates a significant decrease from BaPDE treatment alone (P <0.05).