Xanthohumol-induced transient superoxide anion radical formation triggers cancer cells into apoptosis via a mitochondria-mediated mechanism

Oxidative stress and increased release of reactive oxygen species (ROS) are associated with apoptosis induction. Here we report ROS-mediated induction of apoptosis by xanthohumol (XN) from hops. XN at concentrations of 1.6-25 mu M induced an immediate and transient increase in superoxide anion radical (O-2(-center dot)) formation in 3 human cancer cell lines (average +/- SD EC50 of maximum O-2(-center dot) induction=3.1 +/- 0.8 mu M), murine macrophages (EC50=4.0 +/- 0.3 mu M), and BPH-1 benign prostate hyperplasia cells (EC50=4.3 +/- 0.1 mu M), as evidenced by the O-2(-center dot)-specific indicator dihydroethidium. MitoSOX Red costaining and experiments using isolated mouse liver mitochondria (EC50=11.4 +/- 1.8 mu M) confirmed mitochondria as the site of intracellular O-2(-center dot) formation. Antimycin A served as positive control (EC50=12.4 +/- 0.9 mu M). XN-mediated O-2(-center dot) release was significantly reduced in BPH-1 rho(0) cells harboring nonfunctional mitochondria (EC50>25 mu M) and by treatment of BPH-1 cells with vitamin C, N-acetylcysteine (NAC), or the superoxide dismutase mimetic MnTMPyP. In addition, we demonstrated a rapid 15% increase in oxidized glutathione and a dose-dependent overall thiol depletion within 6 h (IC50=24.3 +/- 11 mu M). Respiratory chain complexes I-III were weakly inhibited by XN in bovine heart submitochondrial particles, but electron flux from complex I and II to complex III was significantly inhibited in BPH-1 cells, with IC50 values of 28.1 +/- 2.4 and 24.4 +/- 5.2 mu M, respectively. Within 15 min, intracellular ATP levels were significantly reduced by XN at 12.5 to 50 mu M concentrations (IC50=26.7 +/- 3.7 mu M). Concomitantly, XN treatment caused a rapid breakdown of the mitochondrial membrane potential and the release of cytochrome c, leading to apoptosis induction. Pre- or coincubation with 2 mM NAC and 50 mu M MnTMPyP at various steps increased XN-mediated IC50 values for cytotoxicity in BPH-1 cells from 6.7 +/- 0.2 to 12.2 +/- 0.1 and 41.4 +/- 7.6 mu M, and it confirmed XN-induced O-2(-center dot) as an essential trigger for apoptosis induction. In summary, we have identified mitochondria as a novel cellular target of XN action, resulting in increased O-2(-center dot) production, disruption of cellular redox balance and mitochondrial integrity, and subsequent apoptosis.-Strathmann, J., Klimo, K., Sauer, S. W., Okun, J. G., Prehn, J. H. M., Gerha user, C. Xanthohumol-induced transient superoxide anion radical formation triggers cancer cells into apoptosis via a mitochondria-mediated mechanism. FASEB J. 24, 2938-2950 (2010). www.fasebj.org