Schizophrenia is characterized by affective, cognitive, neuromorphological, and molecular abnormalities that may have a neurodevelopmental origin. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of 854 miRNAs in prefrontal cortical tissue from 100 control, schizophrenic, and bipolar subjects. The cyclic AMP-responsive element binding- and NMDA-regulated microRNA miR-132 was significantly down-regulated in both the schizophrenic discovery cohort and a second, independent set of schizophrenic subjects. Analysis of miR-132 target gene expression in schizophrenia gene-expression microarrays identified 26 genes up-regulated in schizophrenia subjects. Consistent with NMDA-mediated hypofunction observed in schizophrenic subjects, administration of an NMDA antagonist to adult mice results in miR-132 down-regulation in the prefrontal cortex. Furthermore, miR-132 expression in the murine prefrontal cortex exhibits significant developmental regulation and overlaps with critical neurodevelopmental processes during adolescence. Adult prefrontal expression of miR-132 can be down-regulated by pharmacologic inhibition of NMDA receptor signaling during a brief postnatal period. Several key genes, including DNMT3A , GATA2 , and DPYSL3 , are regulated by miR-132 and exhibited altered expression either during normal neurodevelopment or in tissue from adult schizophrenic subjects. Our data suggest miR-132 dysregulation and subsequent abnormal expression of miR-132 target genes contribute to the neurodevelopmental and neuromorphological pathologies present in schizophrenia.

Using quantitative analyses, we identified microRNAs (miRNAs) that were abundantly expressed in visual cortex and that responded to dark rearing and/or monocular deprivation. The most substantially altered miRNA, miR-132, was rapidly upregulated after eye opening and was delayed by dark rearing. In vivo inhibition of miR-132 in mice prevented ocular dominance plasticity in identified neurons following monocular deprivation and affected the maturation of dendritic spines, demonstrating its critical role in the plasticity of visual cortex circuits.

Abstract It is now well accepted that epigenetic alterations contribute to tumorigenesis. DNA methylation has been recognized as an early and possibly initiating event in the development of breast cancer and thus represents a potentially valuable marker for early detection and chemoprevention. In the present study, we aimed to characterize DNA methylation changes associated with the development of breast cancer in the C3(1)/SV40 large T-antigen transgenic mouse model of mammary carcinogenesis. This model has been utilized in various studies to demonstrate chemopreventive efficacy of both natural products as well as pharmacological agents such as green and black tea or the COX-2 inhibitor celecoxib. For DNA methylation analyses, wildtype (wt) and transgenic (tg) mice were sacrificed at 4–24 weeks of age to collect mammary and tumor tissue, respectively. Initially, we selected eight candidate genes know to be silenced by hypermethlytion in human breast cancer: Cadherin1, Cyclin D2, RASSF1a, Timp3, Twist, GSTπ, SMPD3, and GHSR1a. DNA methylation of CpG islands associated with these genes was quantified by EpiTYPER MassARRAY technology based on mass spectrometry. Interestingly, none of these genes was hypermethylated in murine tumors. On average, we detected less than 16% methylation, which was not significantly different between wt and tg tissue and did not change with age. Consequently, we next decided to analyze methylation changes at a genome-wide level, using methylation-specific DNA array analysis after methyl-CpG immunoprecipitation (MCIp). Hypermethylated DNA of age-matched wt and tg animals was enriched by binding to recombinant methyl-CpG-binding domain protein MBD2. Dual-color labeled samples were then hybridized to Agilent murine CpG island microarrays containing oligonucleotide probes for >16.000 CpG islands. Based on data obtained with tissue derived of animals aged 16–24 weeks, we selected genes commonly hypermethylated in tg mice for subsequent validation by MassARRAY analysis. As an example, five genes, Mab21l2, Lyl1, Atp6v1b1, Espn, A930037G, showed significant hypermethylation in a range of 52 to 69% in tumor tissue of mice aged 24 weeks, whereas an average methylation of 6 to 15% was detected in mammary tissue of age-matched wt animals. Interestingly, when we analyzed mammary tissue of mice at increasing age, we observed a gradual increase in hypermethylation in tg vs. wt animals for all selected genes, starting at 12–16 weeks of age even before tumors were detected. These data indicate a potential early role of deregulated DNA methylation in C3(1)/SV40 large T-antigen-induced mammary carcinogenesis. The newly identified genes, which have rarely been investigated in relation to human breast cancer so far, may serve as interesting targets for chemoprevention studies and will be further characterized. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B49.

Abstract Epigenetic silencing of tumor suppressor genes by DNA hypermethylation is now recognized as a hallmark of cancer. Thus, reversal of DNA hypermethylation by demethylating agents and DNA methyltranserase (Dnmt) inhibitors has been extensively investigated. Recently, several studies have shown that natural dietary compounds have the potential to reactivate epigenetically silenced genes by various mechanisms, which might have an impact on chemoprevention strategies. Xanthohumol (XN), a prenylated chalcone from hops, exerts a broad spectrum of chemopreventive actions and inhibits mammary carcinogenesis in vivo. In this study, we report for the first time that epigenetic deregulation of gene expression during carcinogenesis is a novel target of XN action. We demonstrated that XN reactivates a series of candidate genes silenced by DNA promoter hypermethylation in human cancer cell lines. By treating human breast and prostate cancer cell lines MCF-7 and LNCaP with subtoxic XN-concentrations for 48 and 72h, respectively, we observed significant dose-dependent re-expression of glutathione-S-transferase Π (GSTPi), Cyclin D2 and RassF1A mRNA, measured by quantitative RT-PCR. The demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) was used as positive control. To address if the XN-mediated mechanism of gene re-expression involved inhibition of Dnmt activity, we tested XN in an in vitro system using the bacterial methlytransferase M.SssI as an enzyme source. XN at concentrations ranging from 3.13–50 µM dose-dependently inhibited M.SssI activity with a halfmaximal inhibitory concentration of 35.2 µM (p<0.05). Computational modelling of XN docking to Dnmt1 indicates steric interaction with the active site. In addition, in MCF-7 and LNCaP cells treated with XN, expression of Dnmt1, 3a and 3b was inhibited by XN-treatment in a dose-dependent manner, both at the transcriptional (p<0.05) and protein level. Next, demethylating potential of XN was investigated. DNA of XN-treated MCF-7 and LNCaP cells was subjected to quantitative methylation analyis using the Sequenom Massarray platform. Whereas treatment for 72h with 1µM of 5-aza-dC significantly reduced DNA hypermethylation of promoter CpG islands of GSTPi, Cyclin D2 and RassF1A by 10–20% (p<0.05), XN-treatment with 5–10µM did not show general DNA demethylation. However, individual CpG sites in the analyzed regions were strongly demethylated upon XN-treatment. Ongoing studies investigate whether demethylation of these single CpG sites is crucial for active transcription, and whether this can be correlated to induced gene-expression. In conclusion, we demonstrated that XN reactivates the expression of hypermethlyated genes by inhibition of Dnmt activity, expression and demethylation of selected CpG sites, which represents a novel chemopreventive mechanism by XN. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B48.

Abstract 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.

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

Abstract Xanthohumol (XN), a prenylated chalcone from hops, possesses a broad spectrum of chemopreventive actions, including anti-inflammatory, antioxidant, and anti-estrogenic mechanisms, and inhibits tumor growth in vitro and in vivo. This study aimed to investigate the anti-androgenic potential of XN with regard to prostate carcinogenesis. In hormone-depleted, androgen-sensitive LNCaP human prostate cancer cells, XN at a concentration of 10 µM inhibited prostate specific antigen (PSA) secretion induced by 25 nM dihydrotestosterone (DHT) by more than 90% (p<0.001) after 24, 48 and 72 h of incubation. In a concentration range of 0.4 to 50 µM, XN dose-dependently inhibited PSA secretion (p<0.001) with a halfmaximal inhibitory concentration of 3.1 ± 0.32 µM after 48 h of incubation. Also, DHT-PSA-and insulin like growth factor (IGF-1) mRNA expression was dose-dependently inhibited by increasing XN concentrations (p<0.05), measured by quantitative RT-PCR. Western Blot analyses of cytosolic and nuclear protein lysates revealed that XN inhibited DHT-induced nuclear translocation of the androgen receptor (AR). Furthermore, in a yeast AR assay, XN inhibited AR-transcription at concentrations of 10 to 100 µM. Docking calculations of XN to the AR indicate steric interactions between the prenyl moiety of XN and Trp741, which could destabilize the AR C-terminal helix 12 and thus further explain the antagonist activity of XN. To investigate anti-androgenic action of XN in vivo, a Hershberger assay with male orchiectomizedWistar rats was performed. Prostate and seminal vesicle weight gain was stimulated by subcutaneous application of 0.2 mg/kg body weight (bw) testosterone propionate (TP). After 12 days, no significant inhibitory effects of oral XN (10 and 30 mg/kg bw) on prostate weights was observed, whereas the anti-androgen flutamide used as a positive control inhibited the 1.2-fold induction of prostate weights by 78% (p<0.001). However, seminal vesicle weight gain induced by TP was significantly reduced by 56 and 29% by XN treatment (10 and 30 mg/kg bw), respectively, and by 92% by flutamide application. Quantitative RT-PCR analyses of prostate RNA confirmed the anti-androgenic effects of XN in vivo. XN and flutamide significantly reduced the expression of androgen-dependent target genes PSA, AR, IGF-1, prostate binding protein C3, myostatin and tyrosine aminotransferase (p<0.05). Taken together, in the present study XN was identified as a potent novel anti-androgen, which might be of use for future prostate cancer prevention trials. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B79.