Abstract A59: Xanthohumol from hops (Humulus lupulus L.) generates superoxide anion radicals via a mitochondria‐mediated mechanism

Xanthohumol (XN), a polyphenol from hops (Humulus lupulus L.), exerts a broad spectrum of cancer chemopreventive activities, including the induction of apoptosis in human cancer cell lines. We speculated that apoptosis induction involved pro‐oxidant effects at the mitochondrial membrane, leading to uncoupling of the respiratory chain and subsequent membrane collapse. Here, we investigated whether XN has potential to induce reactive oxygen species (ROS). By co‐treatment of benign prostate hyperplasia cells (BPH‐1) with XN and dihydroethidium (DHE), specifically oxidized by superoxide anion radicals (O₂^‐*), we measured an immediate dose‐ and time‐dependent increase in fluorescence, whereas Amplex Red, specific for hydrogen peroxide (H₂O₂), was not oxidized, indicating the formation of O₂^‐*. O₂^‐* formation was significantly inhibited by co‐treatment with the anti‐oxidants ascorbic acid and N‐acetyl cysteine or the superoxide dismutase mimetic MnTMPyP. Fluorescence microscopy images of BPH‐1 stained with MitoSOX Red, specific for mitochondrial O₂^‐*, suggested a mitochodrial origin of O₂^‐* formation, which was confirmed by XN‐mediated induction of O₂^‐* in isolated mitochondria. Furthermore, in BPH‐1‐ρ⁰ (rho‐zero) cells, harboring non‐functional mitochondria, XN‐treatment did not induce O₂^‐*. As one mechanism of XN‐mediated O₂^‐* formation, we hypothesize that XN is oxidized to its respective phenoxylradical by intracellular oxidases in the presence of glutathione (GSH) and traces of H₂O₂, which produces O₂^‐* as side poduct. This was supported by the finding that inhibition of oxidases with diphenylene iodonium (DPI) significantly inhibited XN‐induced O₂^‐* formation. Additionally, we demonstrated a time‐ and dose‐dependent increase in oxidized glutathione (GSSG) and global GSH depletion upon XN‐treatment. Strikingly, within 15min after XN treatment, intracellular ATP production was reduced by 94% and we observed direct inhibition of complexes I – III of the respiratory chain in submitochondrial particles from bovine heart, as well as in BPH‐1. In parallel, the mitochondrial membrane potential broke down already after 10min of XN‐treatment and as a consequence, release of cytochrome c was observed, leading to the induction of apoptosis.

In this study, we demonstrate cancer chemopreventive activity of XN by the induction of O₂^‐*, which disrupts the cellular redox balance and mitochondrial integrity and subsequently triggers cancer cells into apoptosis.

Citation Information: Cancer Prev Res 2010;3(1 Suppl):A59.