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

Abstract

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 microM 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 microM), murine macrophages (EC(50)=4.0+/-0.3 microM), and BPH-1 benign prostate hyperplasia cells (EC(50)=4.3+/-0.1 microM), 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 microM) confirmed mitochondria as the site of intracellular O(2)(-*) formation. Antimycin A served as positive control (EC(50)=12.4+/-0.9 microM). XN-mediated O(2)(-*) release was significantly reduced in BPH-1 rho(0) cells harboring nonfunctional mitochondria (EC(50)>25 microM) 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 microM). 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 microM, respectively. Within 15 min, intracellular ATP levels were significantly reduced by XN at 12.5 to 50 microM concentrations (IC(50)=26.7+/-3.7 microM). 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 microM 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 microM, 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.