Black raspberry components inhibit proliferation, induce apoptosis, and modulate gene expression in rat esophageal epithelial cells

Abstract

We have shown that a diet containing freeze-dried black raspberries (BRB) inhibits the development of chemically induced cancer in the rat esophagus. To provide insights into possible mechanisms by which BRB inhibit esophageal carcinogenesis, we evaluated an ethanol (EtOH) extract of BRB, and two component anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) in BRB, for their effects on growth, apoptosis, and gene expression in rat esophageal epithelial cell lines. The EtOH extract and both anthocyanins selectively caused significant growth inhibition and induction of apoptosis in a highly tumorigenic cell line (RE-149 DHD) but not in a weakly tumorigenic line (RE-149). The uptake of anthocyanins from the EtOH extract into RE-149 DHD cells far exceeded their uptake into RE-149 cells, which may have accounted for the selective effects of the extract on growth and apoptosis of RE-149 DHD cells. The growth inhibitory and proapoptotic effects were enhanced by the daily addition of the EtOH extract and the anthocyanins to the medium. Interestingly, the EtOH extract did not alter cyclooxygenase-2 (COX-2) and nitric oxide synthase (i-NOS) expression in RE-149 DHD cells, whereas both anthocyanins downregulated the expressions of these genes. This differential effect may have been related to the relative amounts of anthocyanins in the extract vs. when they were added individually to the medium. We conclude that the selective effects of the EtOH extract on growth and apoptosis of highly tumorigenic rat esophageal epithelial cells in vitro may be due to preferential uptake and retention of its component anthocyanins, and this may also be responsible for the greater inhibitory effects of freeze-dried whole berries on tumor cells in vivo.

Figure 1

The addition of 30 units of catalase to the medium does not reduce the inhibitory effect of the 100 µg EtOH extract on the growth of RE-149 DHD cells. The increased antiproliferative activity of the EtOH extract in the presence of catalase was not significant compared to the antiproliferative activity of the extract alone (p>0.05).

Figure 2

100 µg EtOH extract/ml induced caspase 3 and 7 activities in RE-149 DHD cells (by 38%) but not in RE-149 cells, when changing the medium on the 3^rd day (A). Adding fresh extract daily increased caspase 3 and 7 activities in RE-149 DHD cells by 51% (B). *p<0.05.

Figure 3

50 µg cyanidin-3-O-rutinoside/ml induced caspase 3 and 7 activities in RE-149 DHD cells (by 23%) but not in RE-149 cells. * p<0.05.

Figure 4

(A). Uptake of anthocyanins at different time points by RE-149 DHD cells treated with 100 µg EtOH extract/ml as measured by HPLC-MS/MS. (B). Uptake of anthocyanins at different time points by RE-149 cells treated with 100 µg EtOH extract/ml as measured by HPLC-MS/MS. (C). Degradation of anthocyanins in medium containing 100 µg EtOH extract/ml and no cells.

Figure 5

100 µg EtOH extract/ml induces the expression of caspase 3 mRNA (A) and caspase 7 mRNA (B) in RE-149 DHD cells. * p<0.05

Figure 6

Effects of 4 days of 100 µg EtOH extract/ml, 50 µg cyanidin-3-O-glucoside/ml and 50 µg cyanidin-3-O-rutinoside treatment on COX-2 (A) and i-NOS (B) gene expression in RE-149 DHD cells. * p<0.05