Clarissa Gerhäuser

Glucosinolates in Brassica vegetables: The influence of the food supply chain on intake, bioavailability and human health

Glucosinolates (GLSs) are found in Brassica vegetables. Examples of these sources include cabbage, Brussels sprouts, broccoli, cauliflower and various root vegetables (e.g. radish and turnip). A number of epidemiological studies have identified an inverse association between consumption of these vegetables and the risk of colon and rectal cancer. Animal studies have shown changes in enzyme activities and DNA damage resulting from consumption of Brassica vegetables or isothiocyanates, the breakdown products (BDP) of GLSs in the body. Mechanistic studies have begun to identify the ways in which the compounds may exert their protective action but the relevance of these studies to protective effects in the human alimentary tract is as yet unproven. In vitro studies with a number of specific isothiocyanates have suggested mechanisms that might be the basis of their chemoprotective effects. The concentration and composition of the GLSs in different plants, but also within a plant (e.g. in the seeds, roots or leaves), can vary greatly and also changes during plant development. Furthermore, the effects of various factors in the supply chain of Brassica vegetables including breeding, cultivation, storage and processing on intake and bioavailability of GLSs are extensively discussed in this paper.

Mechanism-based in vitro screening of potential cancer chemopreventive agents

Identification and use of effective cancer chemopreventive agents have become an important issue in public health-related research. For identification of potential cancer chemopreventive constituents we have set up a battery of cell- and enzyme-based in vitro marker systems relevant for prevention of carcinogenesis in vivo. These systems include modulation of drug metabolism (inhibition of Cyp1A activity, induction of NAD(P)H:quinone reductase (QR) activity in Hepa1c1c7 murine hepatoma cell culture), determination of radical scavenging (DPPH scavenging) and antioxidant effects (scavenging of superoxide anion-, hydroxyl- and peroxyl-radicals), anti-inflammatory mechanisms (inhibition of lipopolysaccharide (LPS)-mediated nitric oxide (NO) generation by inducible NO synthase (iNOS) in Raw 264.7 murine macrophages, cyclooxygenase-1 (Cox-1) inhibition), and anti-tumor promoting activities (inhibition of phorbol ester-induced ornithine decarboxylase (ODC) activity in 308 murine keratinocytes). We have tested a series of known chemopreventive substances belonging to several structural classes as reference compounds for the identification of novel chemopreventive agents or mechanisms. These include organosulfur compounds (phenethylisothiocyanate (PEITC), diallylsulfide, diallyldisulfide), terpenes (limonene, perillyl alcohol, oleanolic acid, 18-beta-glycyrrhetinic acid), short-chain fatty acids (sodium butyrate), indoles (indole-3-carbinol), isoflavonoids (quercetin, silymarin, genistein), catechins ((-)-epigallocatechin gallate (EGCG)), simple phenols (ellagic acid, resveratrol, piceatannol, curcumin), pharmaceutical agents (piroxicam, acetylsalicylic acid, tamoxifen), and vitamins/derivatives (ascorbic acid, Trolox). We confirmed known chemopreventive mechanisms of these compounds. Additionally, we could demonstrate the usefulness of our approach by identification of hitherto unknown mechanisms of selected agents. As an example, we detected anti-inflammatory properties of PEITC, based on NF-kappaB-mediated inhibition of NO production. Further, PEITC inhibited phorbol ester-induced superoxide anion radical production in granulocytes, and ODC induction in the 308 cell line. These mechanisms might contribute to the chemopreventive potential of PEITC.

Genomic Deletion of PTEN Is Associated with Tumor Progression and Early PSA Recurrence in ERG Fusion-Positive and Fusion-Negative Prostate Cancer

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is often altered in prostate cancer. To determine the prevalence and clinical significance of the different mechanisms of PTEN inactivation, we analyzed PTEN deletions in TMAs containing 4699 hormone-naïve and 57 hormone-refractory prostate cancers using fluorescence in situ hybridization analysis. PTEN mutations and methylation were analyzed in subsets of 149 and 34 tumors, respectively. PTEN deletions were present in 20.2% (458/2266) of prostate cancers, including 8.1% heterozygous and 12.1% homozygous deletions, and were linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), presence of lymph node metastasis (P = 0.0002), hormone-refractory disease (P < 0.0001), presence of ERG gene fusion (P < 0.0001), and nuclear p53 accumulation (P < 0.0001). PTEN deletions were also associated with early prostate-specific antigen recurrence in univariate (P < 0.0001) and multivariate (P = 0.0158) analyses. The prognostic impact of PTEN deletion was seen in both ERG fusion-positive and ERG fusion-negative tumors. PTEN mutations were found in 4 (12.9%) of 31 cancers with heterozygous PTEN deletions but in only 1 (2%) of 59 cancers without PTEN deletion (P = 0.027). Aberrant PTEN promoter methylation was not detected in 34 tumors. The results of this study demonstrate that biallelic PTEN inactivation, by either homozygous deletion or deletion of one allele and mutation of the other, occurs in most PTEN-defective cancers and characterizes a particularly aggressive subset of metastatic and hormone-refractory prostate cancers.

Cancer Chemopreventive Potential of Apples, Apple Juice, and Apple Components

Apples ( MALUS sp., Rosaceae) are a rich source of nutrient as well as non-nutrient components and contain high levels of polyphenols and other phytochemicals. Main structural classes of apple constituents include hydroxycinnamic acids, dihydrochalcones, flavonols (quercetin glycosides), catechins and oligomeric procyanidins, as well as triterpenoids in apple peel and anthocyanins in red apples. Several lines of evidence suggest that apples and apple products possess a wide range of biological activities which may contribute to health beneficial effects against cardiovascular disease, asthma and pulmonary dysfunction, diabetes, obesity, and cancer (reviewed by Boyer and Liu, Nutr J 2004). The present review will summarize the current knowledge on potential cancer preventive effects of apples, apple juice and apple extracts (jointly designated as apple products). In brief, apple extracts and components, especially oligomeric procyanidins, have been shown to influence multiple mechanisms relevant for cancer prevention in IN VITRO studies. These include antimutagenic activity, modulation of carcinogen metabolism, antioxidant activity, anti-inflammatory mechanisms, modulation of signal transduction pathways, antiproliferative and apoptosis-inducing activity, as well as novel mechanisms on epigenetic events and innate immunity. Apple products have been shown to prevent skin, mammary and colon carcinogenesis in animal models. Epidemiological observations indicate that regular consumption of one or more apples a day may reduce the risk for lung and colon cancer.

Inhibition of angiogenesis and endothelial cell functions are novel sulforaphane-mediated mechanisms in chemoprevention.

Sulforaphane, an aliphatic isothiocyanate, is a known cancer chemopreventive agent. Aiming to investigate antiangiogenic potential of sulforaphane, we here report a potent decrease of newly formed microcapillaries in a human in vitro antiangiogenesis model, with an IC50 of 0.08 μmol/L. The effects of sulforaphane on endothelial cell functions essential for angiogenesis were investigated in HMEC-1, an immortalized human microvascular endothelial cell line. Molecular signaling pathways leading to activation of endothelial cell proliferation and degradation of the basement membrane were analyzed by reverse transcription-PCR. Sulforaphane showed time- and concentration-dependent inhibitory effects on hypoxia-induced mRNA expression of vascular endothelial growth factor and two angiogenesis-associated transcription factors, hypoxia-inducible factor-1α and c-Myc, in a concentration range of 0.8 to 25 μmol/L. In addition, the expression of the vascular endothelial growth factor receptor KDR/flk-1 was inhibited by sulforaphane at the transcriptional level. Sulforaphane could also affect basement membrane integrity, as it suppressed transcription of the predominant endothelial collagenase matrix metalloproteinase-2 and its tissue inhibitor of metalloproteinase-2. Migration of HMEC-1 cells in a wound healing assay was effectively prevented by sulforaphane at submicromolar concentrations, and we determined an IC50 of 0.69 μmol/L. In addition, within 6 hours of incubation, sulforaphane inhibited tube formation of HMEC-1 cells on basement membrane matrix at 0.1, 1, and 10 μmol/L concentrations. These effects were not due to inhibition of HMEC-1 cell proliferation; however, after 72 hours of incubation, sulforaphane nonselectively reduced HMEC-1 cell growth with an IC50 of 11.3 μmol/L. In conclusion, we have shown that sulforaphane interferes with all essential steps of neovascularization from proangiogenic signaling and basement membrane integrity to endothelial cell proliferation, migration, and tube formation. These novel antiangiogenic activities of sulforaphane are likely to contribute to its cancer chemopreventive and therapeutic potential. [Mol Cancer Ther 2006;5(3):575–85]

Cancer Chemoprevention and Nutri-Epigenetics: State of the Art and Future Challenges

The term “epigenetics” refers to modifications in gene expression caused by heritable, but potentially reversible, changes in DNA methylation and chromatin structure. Epigenetic alterations have been identified as promising new targets for cancer prevention strategies as they occur early during carcinogenesis and represent potentially initiating events for cancer development. Over the past few years, nutri-epigenetics – the influence of dietary components on mechanisms influencing the epigenome – has emerged as an exciting new field in current epigenetic research. During carcinogenesis, major cellular functions and pathways, including drug metabolism, cell cycle regulation, potential to repair DNA damage or to induce apoptosis, response to inflammatory stimuli, cell signalling, and cell growth control and differentiation become deregulated. Recent evidence now indicates that epigenetic alterations contribute to these cellular defects, for example epigenetic silencing of detoxifying enzymes, tumor suppressor genes, cell cycle regulators, apoptosis-inducing and DNA repair genes, nuclear receptors, signal transducers and transcription factors by promoter methylation, and modifications of histones and non-histone proteins such as p53, NF-κB, and the chaperone HSP90 by acetylation or methylation.

Rotenoids mediate potent cancer chemopreventive activity through transcriptional regulation of ornithine decarboxylase

For the discovery of new cancer chemopreventive agents, we have studied the potential of plant extracts to inhibit phorbol ester-induced ornithine decarboxylase (ODC) activity in cell culture. Four active rotenoids were obtained from the African plant Mundulea sericea (Leguminosae). These isolates were highly potent when evaluated for inhibition of chemically induced preneoplastic lesions in mammary organ culture and inhibition of papillomas in the two-stage mouse skin model, and they appear to function by a unique mechanism at the level of ODC messenger RNA expression. Based on our findings, rotenoids can be regarded as promising new chemopreventive or anticancer agents.

Pan-cancer patterns of DNA methylation

The comparison of DNA methylation patterns across cancer types (pan-cancer methylome analyses) has revealed distinct subgroups of tumors that share similar methylation patterns. Integration of these data with the wealth of information derived from cancer genome profiling studies performed by large international consortia has provided novel insights into the cellular aberrations that contribute to cancer development. There is evidence that genetic mutations in epigenetic regulators (such as DNMT3, IDH1/2 or H3.3) mediate or contribute to these patterns, although a unifying molecular mechanism underlying the global alterations of DNA methylation has largely been elusive. Knowledge gained from pan-cancer methylome analyses will aid the development of diagnostic and prognostic biomarkers, improve patient stratification and the discovery of novel druggable targets for therapy, and will generate hypotheses for innovative clinical trial designs based on methylation subgroups rather than on cancer subtypes. In this review, we discuss recent advances in the global profiling of tumor genomes for aberrant DNA methylation and the integration of these data with cancer genome profiling data, highlight potential mechanisms leading to different methylation subgroups, and show how this information can be used in basic research and for translational applications. A remaining challenge is to experimentally prove the functional link between observed pan-cancer methylation patterns, the associated genetic aberrations, and their relevance for the development of cancer.

In vitro chemopreventive potential of fucophlorethols from the brown alga Fucus vesiculosus L. by anti-oxidant activity and inhibition of selected cytochrome P450 enzymes.

Within a project focusing on the chemopreventive potential of algal phenols, two phloroglucinol derivatives, belonging to the class of fucophlorethols, and the known fucotriphlorethol A were obtained from the ethanolic extract of the brown alga Fucus vesiculosus L. The compounds trifucodiphlorethol A and trifucotriphlorethol A are composed of six and seven units of phloroglucinol, respectively. The compounds were examined for their cancer chemopreventive potential, in comparison with the monomer phloroglucinol. Trifucodiphlorethol A, trifucotriphlorethol A as well as fucotriphlorethol A were identified as strong radical scavengers, with IC(50) values for scavenging of 1,1-diphenyl-2 picrylhydrazyl radicals (DPPH) in the range of 10.0-14.4 microg/ml. All three compounds potently scavenged peroxyl radicals in the oxygen radical absorbance capacity (ORAC) assay. In addition, the compounds were shown to inhibit cytochrome P450 1A activity with IC(50) values in the range of 17.9-33 microg/ml, and aromatase (Cyp19) activity with IC(50) values in the range of 1.2-5.6 microg/ml.

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 (O2−·) formation in 3 human cancer cell lines (average ±sd EC50 of maximum O2−· induction=3.1 ±0.8 µM), murine macrophages (EC50=4.0±0.3 µM), and BPH‐1 benign prostate hyperplasia cells (EC50=4.3±0.1 µM), as evidenced by the O2−· ‐specific indicator dihydroethidium. MitoSOX Red costaining and experiments using isolated mouse liver mitochondria (EC50=11.4±1.8 µM) confirmed mitochondria as the site of intracellular O2−· formation. Antimycin A served as positive control (EC50=12.4±0.9 µM). XN‐mediated O2−· release was significantly reduced in BPH‐1 ρ0 cells harboring nonfunctional mitochondria (EC50>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 (IC50 = 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 IC50 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 (IC50 = 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 IC50 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 O2−· as an essential trigger for apoptosis induction. In summary, we have identified mitochondria as a novel cellular target of XN action, resulting in increased O2−· 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