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 µM induced an immediate and transient increase in superoxide anion radical (O 2 −· ) formation in 3 human cancer cell lines (average ± sd EC 50 of maximum O 2 −· induction=3.1 ±0.8 µM), murine macrophages (EC 50 =4.0±0.3 µM), and BPH‐1 benign prostate hyperplasia cells (EC 50 =4.3±0.1 µM), as evidenced by the O 2 −· ‐specific indicator dihydroethidium. MitoSOX Red costaining and experiments using isolated mouse liver mitochondria (EC 50 =11.4±1.8 µM) confirmed mitochondria as the site of intracellular O 2 −· formation. Antimycin A served as positive control (EC 50 =12.4±0.9 µM). XN‐mediated O 2 −· release was significantly reduced in BPH‐1 ρ 0 cells harboring nonfunctional mitochondria (EC 50 >25 µM) 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 (IC 50 = 24.3±11 µM). 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 IC 50 values of 28.1 ± 2.4 and 24.4 ± 5.2 µM, respectively. Within 15 min, intracellular ATP levels were significantly reduced by XN at 12.5 to 50 µM concentrations (IC 50 = 26.7 ±3.7 µM). 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 µM MnTMPyP at various steps increased XN‐mediated IC 50 values for cytotoxicity in BPH‐1 cells from 6.7 ± 0.2 to 12.2 ± 0.1 and 41.4 ± 7.6 µM, and it confirmed XN‐induced O 2 −· 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 −· 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., Gerhäuser, 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