Karin Klimo

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.

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

Fractionation of polyphenol‐enriched apple juice extracts to identify constituents with cancer chemopreventive potential

Apples and apple juices are widely consumed and rich sources of phytochemicals. The aim of the present study was to determine which apple constituents contribute to potential chemopreventive activities, using a bioactivity-directed approach. A polyphenol-enriched apple juice extract was fractionated by various techniques. Extract and fractions were tested in a series of test systems indicative of cancer preventive potential. These test systems measured antioxidant effects, modulation of carcinogen metabolism, anti-inflammatory and antihormonal activities, and antiproliferative potential. Regression analyses indicated that 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging potential correlated with the sum of low molecular weight (LMW) antioxidants (including chlorogenic acid, flavan-3-ols, and flavonols) and procyanidins, whereas peroxyl radicals were more effectively scavenged by LMW compounds than by procyanidins. Quercetin aglycone was identified as a potent Cyp1A inhibitor, whereas phloretin and (-)-epicatechin were the most potent cyclooxygenase 1 (Cox-1) inhibitors. Aromatase and Cyp1A inhibitory potential and cytotoxicity toward HCT116 colon cancer cells increased with increasing content in procyanidins. Overall, apple juice constituents belonging to different structural classes have distinct profiles of biological activity in these in vitro test systems. Since carcinogenesis is a complex process, combination of compounds with complementary activities may lead to enhanced preventive effects.

Monodictyochromes A and B, Dimeric Xanthone Derivatives from the Marine Algicolous Fungus Monodictys putredinis

Investigations of the marine-derived fungus Monodictys putredinis led to the isolation of two novel dimeric chromanones (1, 2) that consist of two uniquely modified xanthone-derived units. The structures were elucidated by extensive spectroscopic measurements including NOE experiments and CD analysis to deduce the configuration. The compounds (1, 2) were examined for their cancer chemopreventive potential and shown to inhibit cytochrome P450 1A activity with IC(50) values of 5.3 and 7.5 μM, respectively. In addition, both compounds displayed moderate activity as inducers of NAD(P)H:quinone reductase (QR) in cultured mouse Hepa 1c1c7 cells, with CD values (concentration required to double the specific activity of QR) of 22.1 and 24.8 μM, respectively. Compound 1 was slightly less potent than compound 2 in inhibiting aromatase activity, with IC(50) values of 24.4 and 16.5 μM.

Noduliprevenone: A Novel Heterodimeric Chromanone with Cancer Chemopreventive Potential

Chemoprevention is considered a new strategy in fighting diverse types of cancer. To influence cancer development and progression, signaling pathways, for example, enzymes like cyclooxygenase-2, transcription factors like NF-kB, and several metabolic processes leading to activation or deactivation of carcinogenic agents, are envisaged as promising approaches. Cytochrome P450 (CYP) enzymes activate xenobiotics and thus contribute to the generation of potent carcinogens. Accordingly, inhibition of cytochrome P450 isoforms, such as CYP1A enzymes, which play an important role in polycyclic aromatic hydrocarbon (PAH) induced cancer forms, is advantageous for chemoprevention. In contrast, the induction of phase II enzymes inactivates carcinogens and accelerates their renal or biliar elimination, forming conjugates with polar ligands, such as glutathione, glucuronic acid, and acetic or sulfuric acid. NAD(P)H:quinone reductase (QR) contributes in detoxification by catalysing the two-electronaccepted reduction of quinones to their corresponding, lowmutagenic hydroquinones and is therefore often used as biomarker. Accordingly, the aim of cancer chemopreventive strategy can include the inhibition of phase I enzymes accompanied with an increased phase II metabolism. The fungus Nodulisporium sp. is an endophyte of a Mediterranean alga, and produces the novel polyketide noduliprevenone (1), an unprecedented structure consisting of two

Identification of 3-hydroxy-β-damascone and related carotenoid-derived aroma compounds as novel potent inducers of Nrf2-mediated phase 2 response with concomitant anti-inflammatory activity

Structural comparison of apple constituents with known inducers of phase two cytoprotective enzymes led to the identification of 3-hydroxy-beta-damascone and related carotenoid derived aroma compounds as potent inducers of NAD(P)H:quinone reductase (QR) activity. Damascone-related compounds were found to be more potent inducers than ionone derivatives, with CD values (concentrations required to double the specific activity of QR in Hepa1c1c7 cell culture) in the range of 1.0-5.7 microM. QR induction by 3-hydroxy-beta-damascone was shown to be mediated via transcription factor Nrf2 signaling in transient transfection experiments. We further identified aroma compounds as potent inhibitors of LPS-induced inducible nitric oxide synthase activity in Raw 264.7 cell culture. Again, damascone derivatives were most potent with half-maximal inhibitory concentration values of 1.8-7.9 microM. These results reveal previously unrecognized cancer chemopreventive potential of aroma compounds such as beta-damascenone, 3-hydroxy-beta-damascone, and related substances, which may contribute to the cancer protective efficacy of apple products and other dietary sources in animal models.

Synthesis of Resveratrol Derivatives and In Vitro Screening for Potential Cancer Chemopreventive Activities.

New resveratrol (trans‐3,4’,5‐trihydroxystilbene) analogs were synthesized and screened for their in vitro cancer chemopreventive potential using various bioassays relevant for the prevention of carcinogenesis in humans: two assays to detect modulators of carcinogen metabolism (Cyp1A inhibition; determination of NAD(P)H/quinone reductase (QR) activity), three assays to identify radical scavenging and antioxidant properties (DPPH, ORAC, superoxide anion radicals in differentiated HL‐60 cells), four assays to determine anti‐inflammatory and anti‐hormonal effects (iNOS, Cox‐1 and aromatase inhibition, anti‐estrogenic potential). 3,4′,5‐Tri‐O‐methyl resveratrol 1a was about sevenfold more active than resveratrol in inhibiting Cyp1A activity, it was a potent inducer of QR activity, and it showed pure anti‐estrogenic activity (whereas resveratrol is a known mixed estrogen (ant)agonist with both estrogenic and anti‐estrogenic properties). Dual estrogen ant‐/agonist activity was restored in the mono‐O‐benzyl‐substituted derivatives 4b (4′‐O‐benzyl resveratrol) and 5b (3‐O‐benzyl resveratrol). With respect to aromatase inhibition (Cyp19), which provided the highest number of actives, the benzyl‐substituted series was more potent than the methyl‐substituted derivatives of resveratrol, and 3‐O‐benzyl resveratrol 5b was about eightfold more active than resveratrol. Overall, 3,4′,5‐tri‐O‐pivaloyl resveratrol oxide 7c was identified as a potent inducer of phase 2 enzymes concomitant with inhibition of LPS‐mediated iNOS induction.

Abstract A59: Xanthohumol from hops (Humulus lupulus L.) generates superoxide anion radicals via a mitochondria‐mediated mechanism

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 (O 2 ‐* ), we measured an immediate dose‐ and time‐dependent increase in fluorescence, whereas Amplex Red, specific for hydrogen peroxide (H 2 O 2 ), was not oxidized, indicating the formation of O 2 ‐* . O 2 ‐* 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 O 2 ‐* , suggested a mitochodrial origin of O 2 ‐* formation, which was confirmed by XN‐mediated induction of O 2 ‐* in isolated mitochondria. Furthermore, in BPH‐1‐ρ 0 (rho‐zero) cells, harboring non‐functional mitochondria, XN‐treatment did not induce O 2 ‐* . As one mechanism of XN‐mediated O 2 ‐* formation, we hypothesize that XN is oxidized to its respective phenoxylradical by intracellular oxidases in the presence of glutathione (GSH) and traces of H 2 O 2 , which produces O 2 ‐* as side poduct. This was supported by the finding that inhibition of oxidases with diphenylene iodonium (DPI) significantly inhibited XN‐induced O 2 ‐* 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 O 2 ‐* , 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.