| Anthocyanins are polyphenols that can prevent colon cancer. The compounds are metabolized by microflora in the large intestine to phenolic acids and aldehydes, and the health benefits of consuming foods rich in anthocyanins could be due mostly to the effects of these metabolites. In this work, an anthocyanin extract from Cabernet Sauvignon grapes was incubated in the large intestinal contents of freshly slaughtered pigs. This pig is a good model for human gut metabolism, as pig and human gut flora are similar. Anthocyanins and their metabolites were measured by LC-ESI-MS. After 6 h, anthocyanins were no longer detected and three metabolites were identified as 3- O-methylgallic acid, syringic acid, and 2,4,6-trihydroxybenzaldehyde (THBA). All six major anthocyanin metabolites, including gallic acid, were then incubated with human colon cancer cells (Caco-2) to determine effects on cell proliferation, cytotoxicity, and induction of apoptosis. Gallic acid, 3-O-methylgallic acid, and THBA inhibited cell proliferation, and had low cytotoxicity. In addition, these same treatments degraded quickly in growth media, suggesting the involvement of subsequent oxidation products in the reduction of cell viability. To understand the underlying mechanisms, we investigated the capacity of these three anthocyanin metabolites to modulate the cell cycle, and to induce apoptotic cell death. Caco-2 cells were incubated with gallic acid, 3-O-methylgallic acid, and THBA. THBA reduced cell viability at the highest treatment concentration (100 &mgr;M). Gallic acid and 3-O-methylgallic acid induced a time and dose-dependent decrease in cell viability. The three metabolites caused an arrest in the G0/G1 phase of the cell cycle, inhibiting cell proliferation. The three metabolites activated caspase-3. However, only gallic acid and 3-O-methylgallic acid increased the levels of mono- and oligonucleosomes within the cytoplasm and caused apoptotic nuclear morphologic changes (Hoescht staining). All three metabolites led to a decrease in NF-κB-DNA binding, which was not related to generation of hydrogen peroxide. In conclusion, the results indicate that the basis of the effects appear to be anti-proliferative and an induction of apoptosis for 3-O-methylgallic acid and gallic acid, and mostly anti-proliferative for THBA. The inhibition of transcription factor NF-κ&Bgr;, which promotes cell proliferation and survival, can underlie the observed effects. |
