Xanthohumol isolated from Humulus lupulus Inhibits menadione-induced DNA damage through induction of quinone reductase

Abstract

The female parts of hops (Humulus lupulus L.) show estrogenic effects as well as cancer chemopreventive potential. We analyzed the chemopreventive mechanism of hops by studying its antioxidative activities and its effect on the detoxification of a potentially toxic quinone (menadione). The detoxification enzyme quinone reductase [(NAD(P)H:quinone oxidoreductase, QR] protects against quinone-induced toxicity and has been used as a marker in cancer chemoprevention studies. Although the hop extract was only a weak quencher of free radicals formed from 1,1-diphenyl-2-picrylhydrazyl, it demonstrated strong QR induction in Hepa 1c1c7 cells. In addition, compounds isolated from hops including xanthohumol (XH) and 8-prenylnaringenin were tested for QR induction. Among these, XH was the most effective at inducing QR with a concentration required to double the specific activity of QR (CD value) of 1.7 +/- 0.7 microM. In addition, pretreatment of Hepa1c1c7 cells with XH significantly inhibited menadione-induced DNA single-strand breaks. The QR inhibitor dicumarol reversed the protective effect of XH against menadione-induced DNA damage. Because the expression of QR and other detoxifying enzymes is known to be upregulated by binding of the transcription factor Nrf2 to the antioxidant response element (ARE), the reporter activity mediated by ARE in HepG2-ARE-C8 cells was investigated after incubation with XH for 24 h. Under these conditions, XH increased ARE reporter activity in a dose-dependent manner. One mechanism by which XH might induce QR could be through interaction with Keap1, which sequesters Nrf2 in the cytoplasm, so that it cannot activate the ARE. Using LC-MS-MS, we demonstrated that XH alkylates human Keap1 protein, most likely on a subset of the 27 cysteines of Keap1. This suggests that XH induces QR by covalently modifying the Keap1 protein. Therefore, XH and hops dietary supplements might function as chemopreventive agents, through induction of detoxification enzymes such as QR.

Figure 1.

Chemical structures of isolated compounds from H. lupulus L. (30).

Figure 2.

QR activity of XH isolated from hops was evaluated using Hepa 1c1c7 murine hepatoma cells. After the cells were treated with XH for 48 h (■), the induction of QR was determined. In parallel, cells (▴) were preincubated with XH for 48 h prior to exposure to dicumarol (15 μM) for 15 min, and subsequently, the QR activity was measured. Each value represents the average fold induction of three experiments performed independently in duplicate ± SD.

Figure 3.

Cytotoxicity of XH was examined in the CV assay. The cells were treated with XH for 48 h. Each value represents the average of three experiments performed independently in duplicate ± SD.

Figure 4.

Inhibitory effect of XH against DNA single-strand breaks induced by menadione was examined using the comet assay. Hepa 1c1c7 mouse hepatoma cells were preincubated with XH for 48 h prior to menadione (10 μM) exposure for 30 min (■), or cells were treated with XH and menadione at the same time for 30 min (▴) before the comet assay was conducted. Each value is the average of triplicate measurements ± SD.

Figure 5.

Hepa 1c1c7 mouse hepatoma cells were preincubated with XH (4 μM) for 48 h prior to exposure to dicumarol (15 μM) for 15 min. DNA single-strand breaks were induced by 10 μM menadione treatment for 30 min. Each value is the average of triplicate measurements ± SD. Means are significantly different (P < 0.05, t-test).

Figure 6.

Induction of the ARE-luciferase reporter gene by XH. Stably transfected HepG2-ARE-C8 cells (54) were plated in six well plates at a density of 1 × 10⁵ cells/mL and incubated overnight. Cells were stimulated with different concentrations of XH, BF as a positive control (40 μg/mL, 6.32 ± 0.98-fold induction), or with DMSO as a negative control. Cells were harvested 24 h after treatment. Luciferase activity was determined and normalized by protein determination. The data were obtained from three separate experiments and expressed as fold induction as compared to the control (DMSO-treated cells) ± SD.

Figure 7.

(A) Positive MALDI-TOF mass spectra of Keap1 (11 μM) modified by incubation with different concentrations of XH. (B) Number of modifications of Keap1 by XH at different ratios of XH to Keap1.

Scheme 1.

Redox Cycling of Menadione Inducing Oxidative Damage or Detoxification by QR^{a a} Mechanism of chemoprevention: 1, antioxidants; 2, quinone reductase.

Scheme 2.

Proposed Mechanism of QR Induction by XH through the Keap1–Nrf2 Pathway^{a a} Besides Keap1, the mitogen-activated protein kinase (MAPK) (53, 54), the protein kinase C (PKC) (55), and the phosphatidylinositol 3-kinase (PI3K) pathway (56, 57) play a role in the regulation of detoxification enzymes (17). PKC phosphorylation of serine-40 in Nrf2 is necessary for Nrf2 release from Keap1 (55). Within the nucleus, Nrf2 binds to the ARE as a heterodimer with either small Maf proteins, FosB, c-Jun, or JunD (46, 58). These proteins are omitted in the scheme for clarity.