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, 65 (1), 118-25

Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes Through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells

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Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes Through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells

Li-Shu Wang et al. Nutr Cancer.

Abstract

We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 μg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of β-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers.

Figures

FIG. 1.
FIG. 1.
Black raspberry-derived anthocyanins (ACs) suppress DNMT1 and DNMT3B. A: Total DNMT activity in nuclear extracts from human colon cancer cells, HCT116, Caco2, and SW480, treated with ACs at 0.5, 5, and 25 μg/ml for 3 days was decreased. B: In a cell free in vitro inhibition assay, ACs inhibit DNMT1 and DNMT3B activities. C: Three days treatment with 25 μg/ml ACs decrease protein expression of DNMT1 and DNMT3B in nuclear extracts from all 3 lines. * P < 0.05.
FIG. 2.
FIG. 2.
Cellular uptake of anthocyanins (ACs) and their colocalization with DNMT proteins in HCT116 cells. A: ACs differentially localize with DNMT1 and DNMT3B in HCT116 cells. B: ACs inhibit c-Myc mRNA expression in HCT116, Caco2 and SW480 cells. C: c-Myc and DNMT3B do not bind with one another in HCT116 cells. (Continued) (Color figure available online).
FIG. 3.
FIG. 3.
Differential responses of human colon cancer cells to anthocyanins (ACs)-induced demethylation. ACs (25 μg/ml, 3 days) demethylate the promoter sequence of CDKN2A in HCT116 and SW480 cells (A), of SFRP5 in Caco2 cells (B), and of SFRP2 (C) and WIF1 (D) in HCT116, Caco2, and SW480 cells. ACs do not induce genome-wide methylation changes in all 3 lines as measured by the LINE-1 repetitive element (E). Ctrl = control. * P < 0.05.
FIG. 4.
FIG. 4.
Effects of anthocyanins (ACs) on mRNA expression of tumor suppressor genes. ACs do not alter the mRNA expression of CDKN2A in HCT116, Caco2, and SW480 cells (A). ACs increase the mRNA expression of SFRP5 in HCT116 cells (B) and of SFRP2 in all 3 cell lines (C). ACs increase WIF1 mRNA expression in HCT116 and SW480 cells (D). * P < 0.05.
FIG. 5.
FIG. 5.
Anthocyanins (ACs) decreased the mRNA expression of β-catenin in HCT116 and SW480 cells (A). This is accompanied by decreased cell proliferation (B) and increased apoptosis (C). * P < 0.05.

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