Investigation of the role of dietary flavonoids on cell death: evidence to support a non‐apoptotic mechanism

Abstract

Flavonoids are polyphenolic compounds widely distributed in plants and have been shown to be cytotoxic to cancer cells. Our lab has previously shown that some flavonoids are able to induce cytotoxicity in human breast cancer cell lines and also able to inhibit caspase activity. Our current study aims to identify the specific molecular pathways required for flavonoid induced cytotoxicity. MDA‐MB‐231 cells, mouse embryonic fibroblasts (MEFs) lacking caspase‐3 and caspase‐7, and primary baby mouse kidney epithelial cells lacking bax and bak were cultured per recommended protocols. Cells were treated with 100 μM flavonoid and cell counts, extracts and/or cytochrome c release were measured at 24, 48, and 72 hours after treatment. Flavonoid‐treated MDA‐MB‐231 cells showed cytochrome c release. We could not detect caspase‐3 or ‐7 cleavage in MDA‐MB‐231 cells at any time point measured. PARP cleavage occurred in MDA‐MB‐231 cells after 6 hours of flavonoid treatment. These results suggest that cytotoxicity in breast cancer cells may involve caspase‐independent mechanisms. To test this, we treated caspase‐deficient MEFs with various flavonoids compared to wild‐type controls and performed cell counts. Flavonoids induced cytotoxicity in caspase‐deficient MEFs similar to wild‐type controls. Finally, we have looked at flavonoid induced cytotoxicity in bax/bak deficient cells and do not see a difference in cytotoxicity compared to wild‐type controls. Taken together, our results suggest that flavonoids are inducing cytotoxicity through a non‐classical apoptotic mechanism which may require the release of cytochrome c but not caspases, bax, or bak.