Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer.

The majority of long noncoding RNAs (lncRNAs) is still poorly characterized with respect to function, interactions with protein-coding genes, and mechanisms that regulate their expression. As for protein-coding RNAs, epigenetic deregulation of lncRNA expression by alterations in DNA methylation might contribute to carcinogenesis. To provide genome-wide information on lncRNAs aberrantly methylated in breast cancer we profiled tumors of the C3(1) SV40TAg mouse model by MCIp-seq (Methylated CpG Immunoprecipitation followed by sequencing). This approach detected 69 lncRNAs differentially methylated between tumor tissue and normal mammary glands, with 26 located in antisense orientation of a protein-coding gene. One of the hypomethylated lncRNAs, 1810019D21Rik (now called Esrp2-antisense (as)) was identified in proximity to the epithelial splicing regulatory protein 2 (Esrp2) that is significantly elevated in C3(1) tumors. ESRPs were shown previously to have a dual role in carcinogenesis. Both gain and loss have been associated with poor prognosis in human cancers, but the mechanisms regulating expression are not known. In-depth analyses indicate that coordinate overexpression of Esrp2 and Esrp2-as inversely correlates with DNA methylation. Luciferase reporter gene assays support co-expression of Esrp2 and the major short Esrp2-as variant from a bidirectional promoter, and transcriptional regulation by methylation of a proximal enhancer. Ultimately, this enhancer-based regulatory mechanism provides a novel explanation for tissue-specific expression differences and upregulation of Esrp2 during carcinogenesis. Knockdown of Esrp2-as reduced Esrp2 protein levels without affecting mRNA expression and resulted in an altered transcriptional profile associated with extracellular matrix (ECM), cell motility and reduced proliferation, whereas overexpression enhanced proliferation. Our findings not only hold true for the murine tumor model, but led to the identification of an unannotated human homolog of Esrp2-as which is significantly upregulated in human breast cancer and associated with poor prognosis.

Nuclear factor kappa B is a molecular target for sulforaphane-mediated anti-inflammatory mechanisms.

Sulforaphane (SFN), an aliphatic isothiocyanate, is a known cancer chemopreventive agent. Aiming to investigate anti-inflammatory mechanisms of SFN, we here report a potent decrease in lipopolysaccharide (LPS)-induced secretion of pro-inflammatory and pro-carcinogenic signaling factors in cultured Raw 264.7 macrophages after SFN treatment, i.e. NO, prostaglandin E(2), and tumor necrosis factor alpha. SFN did not directly interact with NO, nor did it inhibit inducible nitric-oxide synthase enzymatic activity. Western blot analyses revealed time- and dose-dependent reduction of LPS-induced inducible nitric-oxide synthase as well as Cox-2 protein expression, which was suppressed at the transcriptional level. To reveal the target of SFN beyond its anti-inflammatory action, we performed electrophoretic mobility shift assay analyses of transcription factor-DNA binding. Consequently, nuclear factor kappa B (NF-kappa B), a pivotal transcription factor in LPS-stimulated pro-inflammatory response, was identified as the key mediator. SFN selectively reduced DNA binding of NF-kappa B without interfering with LPS-induced degradation of the inhibitor of NF-kappa B nor with nuclear translocation of NF-kappa B. Because SFN can interact with thiol groups by dithiocarbamate formation, it may impair the redox-sensitive DNA binding and transactivation of NF-kappa B. Sulforaphane could either directly inactivate NF-kappa B subunits by binding to essential Cys residues or interact with glutathione or other redox regulators like thioredoxin and Ref-1 relevant for NF-kappa B function. Our data provide novel evidence that anti-inflammatory mechanisms contribute to sulforaphane-mediated cancer chemoprevention.

New lanostanoids from Ganoderma lucidum that induce NAD(P)H:quinone oxidoreductase in cultured hepalcic7 murine hepatoma cells.

Two new lanostanoids were isolated from the basidiocarp of Ganoderma lucidum and were identified as 26,27-dihydroxy-5 alpha-lanosta-7,9(11),24-triene-3,22-dione (1) and 26-hydroxy-5 alpha-lanosta-7,9(11),24-triene-3,22-dione (2) by their respective spectral data. Crude extracts and the isolated compounds were tested for their potential to induce NAD(P)H:quinone oxidoreductase (QR), a phase 2 drug-metabolizing enzyme, as an approach to detect potential cancer chemopreventive activity. Compound 2 doubled the specific activity of QR at a concentration of 3.0 micrograms/ml, whereas compound 1 was significantly less active (1.7-fold induction at 20 micrograms/ml). In addition, both compounds weakly inhibited sheep vesicle cyclooxygenase 1 activity at a test concentration of 40 micrograms/ml.

Amide analogues of trichostatin A as inhibitors of histone deacetylase and inducers of terminal cell differentiation.

Inhibitors of histone deacetylase (HD) bear great potential as new drugs due to their ability to modulate transcription and to induce apoptosis or differentiation in cancer cells. We have described previously analogues of the complex natural HD inhibitors trapoxin B and trichostatin A with activities in the submicromolar range. Here we report structure-activity relationship analyses of further analogues of trichostatin A with respect to in vitro inhibition of maize HD-2 and their ability to induce terminal cell differentiation in Friend leukemic cells. This is the first report that shows the correlation between HD inhibitory activity and action on cancer cells on a larger series of similar compounds. Only the compounds that inhibit HD induce differentiation and/or exert antiproliferative activities in cell culture. Our studies support the use of in vitro systems as screening tools and provide structure-activity relationships that merit further investigation of this interesting target.

Inhibitory effect of munetone, an isoflavonoid, on 12-O-tetradecanoylphorbol 13-acetate-induced ornithine decarboxylase activity.

Starting with an extract derived from the bark of Mundulea sericea Willd. (Leguminosae) that was active in the process of inhibiting 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced ornithine decarboxylase activity (ODC) in cultured mouse epidermal ME 308 cells, the isoflavonoid munetone was isolated and identified as an active principle (IC50 = 46 ng/ml). Topical application of munetone (0.04-5 micromol) to the skin of CD-1 mice 2 h prior to treatment with TPA (10 nmol) resulted in dose-dependent inhibition of epidermal ODC activity. In addition, munetone inhibited TPA-independent c-Myc-induced ODC activity with cultured BALB/c c-MycER cells, as well as 7,12-dimethylbenz[a]anthracene (DMBA)-induced preneoplastic lesion formation in a mouse mammary gland organ culture (MMOC) system. These data suggest the potential of munetone to serve as a cancer chemopreventive agent by virtue of blocking the process of tumor promotion.

Cancer chemopreventive activity mediated by 4'-bromoflavone, a potent inducer of phase II detoxification enzymes.

Induction of phase II enzymes is an important mechanism of chemoprevention. In our search for novel cancer chemopreventive agents, 4'-bromoflavone (4'BF) was found to significantly induce quinone reductase (QR) activity in cultured murine hepatoma 1c1c7 cells (concentration to double activity: 10 nM) and effectively induce the alpha- and mu-isoforms of glutathione S-transferase in cultured H4IIE rat hepatoma cells with no observed toxicity. In short-term dietary studies, 4'BF was also shown to increase QR activity and glutathione levels in rat liver, mammary gland, colon, stomach, and lung in a dose-dependent manner. Induction mediated by 4'BF was bifunctional (induction of both phase I and phase II enzymes) and regulated at the transcriptional level, as revealed by transient transfection studies with plasmid constructs (pDTD-1097CAT, XRE-CAT, and ARE-CAT) and reverse transcription-PCR-based analysis of QR mRNA. In studies conducted with female Sprague Dawley rats, the effects of 4'BF on the relative induction levels of phase I and phase II enzyme activities were investigated in liver and mammary gland. Treatment with 4'BF and 7,12-dimethylbenz[a]anthracene (DMBA) or 4'BF alone did not significantly alter DMBA-induced cytochrome P4501A1 activity (phase I enzyme), but it significantly increased QR activity (phase II enzyme), compared with the DMBA treatment group. In addition, 4'BF was found to be a potent inhibitor of cytochrome P4501A1-mediated ethoxyresorufin-O-deethylase activity, with an IC50 of 0.86 microM. Furthermore, in studies conducted with cultured HepG2 or MCF-7 cells, 4'BF significantly reduced the covalent binding of metabolically activated benzo[a]pyrene to cellular DNA. On the basis of these results, a full-term cancer chemoprevention study was conducted with DMBA-treated female Sprague Dawley rats. Dietary administration of 4'BF (2000 and 4000 mg per kg of diet, from 1 week before to 1 week after DMBA) significantly inhibited the incidence and multiplicity of mammary tumors and greatly increased tumor latency. In summary, 4'BF can be viewed as a relatively simple, readily available, inexpensive compound that is a highly effective cancer chemopreventive agent. The full mechanism of action remains to be defined, but enhancement of detoxification pathways appears to be important.

Activity-guided isolation of constituents of Tephrosia purpurea with the potential to induce the phase II enzyme, quinone reductase.

An isoflavone, 7,4'-dihydroxy-3',5'-dimethoxyisoflavone (1), and a chalcone, (+)-tephropurpurin (2), both novel compounds, as well as six constituents of known structure, (+)-purpurin (3), pongamol (4), lanceolatin B (5), (-)-maackiain (6), (-)-3-hydroxy-4-methoxy-8,9-methylene-dioxypterocarpan (7), and (-)-medicarpin (8), were obtained as active compounds from Tephrosia purpurea, using a bioassay based on the induction of quinone reductase (QR) activity with cultured Hepa 1c1c7 mouse hepatoma cells. Additionally, three inactive compounds of known structure, 3'-methoxydaidzein, desmoxyphyllin B, and 3,9-dihydroxy-8-methoxycoumestan, were isolated and identified. The structure elucidation of compounds 1 and 2 was carried out by spectral data interpretation.

Cancer chemopreventive activity mediated by deguelin, a naturally occurring rotenoid.

Deguelin, a natural product isolated from Mundulea sericea (Leguminosae), was shown previously to mediate strong inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in cell culture and to reduce the formation of preneoplastic lesions when mouse mammary glands were exposed to 7,12-dimethylbenz(a)anthracene. As reported currently, deguelin was synthesized and evaluated for chemopreventive activity in the two-stage 7,12-dimethylbenz(a)anthracene/TPA skin carcinogenesis model with CD-1 mice and in the N-methylnitrosourea mammary carcinogenesis model with Sprague Dawley rats. In the mouse skin study, deguelin reduced tumor incidence from 60% in the control group to 10% in the group treated with a dose of 33 microg, and multiplicity was reduced from 4.2 in the control group to 0.1 in the treatment group. When the dose was increased 10-fold to 330 microg, no tumors were observed in the treatment group. These results correlated with the potential of deguelin to inhibit TPA-induced mouse epidermal ODC activity. When applied topically as a single dose in a time range of 2 h before to 2 h after TPA treatment, deguelin (384 microg) reduced ODC induction by TPA (6.17 microg) by more than 85%. Time course studies indicated that deguelin (33 microg) inhibited TPA (1.17 microg)-induced ODC activity by 70% without affecting the kinetics of induction over a period of 10 h. Complete inhibition of ODC induction was observed at a dose of 330 microg of deguelin. In the rat mammary tumorigenesis study, intragastric administration of 2 or 4 mg of deguelin/kg of body weight daily, 5 days/week, reduced tumor multiplicity from 6.8 tumors/rat in the control group to 5.1 or 3.2 tumors/animal, respectively. At the 4 mg of deguelin/kg of body weight dose level, the tumor latency period was significantly increased. Tumor incidence, however, was unaffected. These data indicate that deguelin exhibits cancer chemopreventive effects in skin and mammary tumorigenesis models and that additional studies are warranted to characterize the cancer chemopreventive or chemotherapeutic potential of this substance more fully.

Regulation of ornithine decarboxylase induction by deguelin, a natural product cancer chemopreventive agent.

Deguelin, a plant-derived rotenoid, mediates potent chemopreventive responses through transcriptional regulation of phorbol ester-induced ornithine decarboxylase (ODC) activity. To explore the mechanism of this effect, the activity of this compound was evaluated with a number of model systems. Using cultured mouse epidermal 308 cells, the steady-state levels of both 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC mRNA and c-fos were decreased by treatment with deguelin. ODC activity was also inhibited by bullatacin and various antimitotic agents (podophyllotoxin, vinblastine, and colchicine), but only deguelin and bullatacin were active as inhibitors of ODC levels in a TPA-independent c-Myc-mediated induction system using cultured BALB/c c-MycER cells. These results suggest that antimicrotubule effects, as mediated by rotenone, for example, are not responsible for inhibitory activity facilitated by deguelin. This was confirmed by use of an in vitro model of tubulin polymerization in which deguelin and a variety of other rotenoids were investigated and found to be inactive. As anticipated, however, NADH dehydrogenase was inhibited by these rotenoids. Moreover, inhibition of this enzyme correlated with a rapid depletion of ATP levels and potential to inhibit either TPA- or c-Myc-induced ODC activity. It therefore seems that deguelin-mediated interference with transient requirements for elevated energy can inhibit the induction of ODC activity and thereby yield a cancer chemopreventive response.

Cancer chemopreventive potential of sulforamate, a novel analogue of sulforaphane that induces phase 2 drug-metabolizing enzymes.

Chemoprevention involves the use of natural or synthetic substances to reduce the risk of developing cancer. Two dietary components capable of mediating chemopreventive activity in animal models by modulation of drug-metabolizing enzymes are sulforaphane, an aliphatic isothiocyanate, and brassinin, an indole-based dithiocarbamate, both found in cruciferous vegetables. We currently report the synthesis and activity of a novel cancer chemopreventive agent, (+/-)-4-methylsulfinyl-1-(S-methyldithiocarbamyl)-butane (trivial name, sulforamate), an aliphatic analogue of brassinin with structural similarities to sulforaphane. This compound was shown to be a monofunctional inducer of NAD(P)H:quinone oxidoreductase [quinone reductase (QR)], a Phase II enzyme, in murine Hepa 1c1c7 cell culture and two mutants thereof. Induction potential was comparable to that observed with sulforaphane (concentration required to double the specific activity of QR, approximately 0.2 microM), but cytotoxicity was reduced by about 3-fold (IC50 approximately 30 microm). In addition, sulforaphane, as well as the analogue, increased glutathione levels about 2-fold in cultured Hepa 1c1c7 cells. Induction of QR was regulated at the transcriptional level. Using Northern blotting techniques, time- and dose-dependent induction of QR mRNA levels were demonstrated in Hepa 1c1c7 cell culture. To further investigate the mechanism of induction, HepG2 human hepatoma cells were transiently transfected with QR-chloramphenicol acetyltransferase plasmid constructs containing various portions of the 5'-region of the QR gene. Sulforaphane and the analogue significantly induced (P < 0.0001) CAT activity at a concentration of 12.5 microM by interaction with the antioxidant responsive element (5-14-fold induction) without interacting with the xenobiotic responsive element. Moreover, both compounds significantly induced mouse mammary QR and glutathione S-transferase activity (feeding of 3 mg/mouse intragastric for 4 days), whereas the elevation of hepatic enzyme activities was less pronounced. Both sulforaphane and the analogue were identified as potent inhibitors of preneoplastic lesion formation in carcinogen-treated mouse mammary glands in organ culture (84 and 78% inhibition at 1 microm, respectively). On the basis of these results, the sulforaphane analogue can be regarded as a readily available promising new cancer chemopreventive agent.

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.

Bryodin, a single-chain ribosome-inactivating protein, selectively inhibits the growth of HIV-1-infected cells and reduces HIV-1 production.

Bryodin, a single-chain ribosome-inactivating protein (RIP) isolated from Bryonia cretica ssp dioica (cucurbitaceae), was found to selectively inhibit the growth of persistently HIV-1-infected T lymphoma cells (KE37/1) and human lung fibroblast when used in concentrations from 2-20 micrograms/ml. Uninfected KE37/1 cells remained unaffected at the same doses of bryodin. In addition, bryodin reduced HIV production in the surviving infected cells. Two isoforms of bryodin were purified by dye ligand chromatography. Both isoforms exerted the growth-inhibiting influence and reduced HIV production. Trichosanthin, another member of the RIP family, had similar inhibitory effects on the growth of HIV-1 infected cells and on HIV-1 production. Bryodin and trichosanthin were effective in about the same dose range. No selective effects for HIV-infected cells were observed with the RIPs gelonin and ricin.