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1.
DNA Repair (Amst) ; 43: 38-47, 2016 07.
Article in English | MEDLINE | ID: mdl-27235629

ABSTRACT

DNA topoisomerase 2 (Top2) poisons, including common anticancer drugs etoposide and doxorubicin kill cancer cells by stabilizing covalent Top2-tyrosyl-DNA 5'-phosphodiester adducts and DNA double-strand breaks (DSBs). Proteolytic degradation of the covalently attached Top2 leaves a 5'-tyrosylated blocked termini which is removed by tyrosyl DNA phosphodiesterase 2 (TDP2), prior to DSB repair through non-homologous end joining (NHEJ). Thus, TDP2 confers resistance of tumor cells to Top2-poisons by repairing such covalent DNA-protein adducts, and its pharmacological inhibition could enhance the efficacy of Top2-poisons. We discovered NSC111041, a selective inhibitor of TDP2, by optimizing a high throughput screening (HTS) assay for TDP2's 5'-tyrosyl phosphodiesterase activity and subsequent validation studies. We found that NSC111041 inhibits TDP2's binding to DNA without getting intercalated into DNA and enhanced etoposide's cytotoxicity synergistically in TDP2-expressing cells but not in TDP2 depleted cells. Furthermore, NSC111041 enhanced formation of etoposide-induced γ-H2AX foci presumably by affecting DSB repair. Immuno-histochemical analysis showed higher TDP2 expression in a sub-set of different type of tumor tissues. These findings underscore the feasibility of clinical use of suitable TDP2 inhibitors in adjuvant therapy with Top2-poisons for a sub-set of cancer patients with high TDP2 expression.


Subject(s)
Aminoquinolines/pharmacology , DNA Repair/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Etoposide/pharmacology , Nuclear Proteins/antagonists & inhibitors , Phosphodiesterase Inhibitors/pharmacology , Topoisomerase Inhibitors/pharmacology , Transcription Factors/antagonists & inhibitors , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Cell Line, Tumor , DNA Adducts/antagonists & inhibitors , DNA Adducts/metabolism , DNA Breaks, Double-Stranded , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Neoplasm/antagonists & inhibitors , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drug Synergism , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/pathology , Gene Expression Regulation, Neoplastic , High-Throughput Screening Assays , Histones/genetics , Histones/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoric Diester Hydrolases , Poly-ADP-Ribose Binding Proteins , Protein Binding , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Toxicology ; 344-346: 7-18, 2016 Feb 17.
Article in English | MEDLINE | ID: mdl-26845733

ABSTRACT

Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H: quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the (32)P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.


Subject(s)
Aristolochic Acids/toxicity , Carcinogens/toxicity , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A2/biosynthesis , DNA Adducts/antagonists & inhibitors , DNA Adducts/biosynthesis , Animals , Enzyme Induction/drug effects , Enzyme Induction/physiology , Male , Rats , Rats, Wistar
3.
Food Chem Toxicol ; 59: 564-71, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23831728

ABSTRACT

Methyleugenol (ME) occurs naturally in a variety of spices, herbs, including basil, and their essential oils. ME induces hepatomas in rodent bioassays following its conversion to a DNA reactive metabolite. In the present study, the basil constituent nevadensin was shown to be able to inhibit SULT-mediated DNA adduct formation in HepG2 cells exposed to the proximate carcinogen 1'-hydroxymethyleugenol in the presence of nevadensin. To investigate possible in vivo implications of SULT inhibition by nevadensin on ME bioactivation, the rat physiologically based kinetic (PBK) model developed in our previous work to describe the dose-dependent bioactivation and detoxification of ME in male rat was combined with the recently developed PBK model describing the dose-dependent kinetics of nevadensin in male rat. The resulting binary ME-nevadensin PBK model was used to predict the possible nevadensin mediated reduction in ME DNA adduct formation and resulting carcinogenicity at the doses of ME used by the NTP carcinogenicity study. Using these data an updated risk assessment using the Margin of Exposure (MOE) approach was performed. The results obtained point at a potential reduction of the cancer risk when rodents are orally exposed to ME within a relevant food matrix containing SULT inhibitors compared to exposure to pure ME.


Subject(s)
Anticarcinogenic Agents/pharmacology , Carcinogens/pharmacokinetics , Eugenol/analogs & derivatives , Flavones/pharmacology , Hepatocytes/metabolism , Models, Biological , Animals , Anticarcinogenic Agents/blood , Anticarcinogenic Agents/metabolism , Anticarcinogenic Agents/pharmacokinetics , Biotransformation/drug effects , Carcinogens/administration & dosage , Carcinogens/metabolism , Carcinogens/toxicity , DNA Adducts/antagonists & inhibitors , DNA Adducts/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/blood , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Eugenol/administration & dosage , Eugenol/metabolism , Eugenol/pharmacokinetics , Eugenol/toxicity , Female , Flavones/blood , Flavones/metabolism , Flavones/pharmacokinetics , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/enzymology , Humans , Male , Rats , Rats, Inbred F344 , Rats, Sprague-Dawley , Risk Assessment , Sulfotransferases/antagonists & inhibitors , Sulfotransferases/metabolism , Tissue Distribution/drug effects
4.
Toxicol Lett ; 208(3): 232-8, 2012 Feb 05.
Article in English | MEDLINE | ID: mdl-22138271

ABSTRACT

This study was designed to investigate the molecular mechanism underlying the chemopreventive effects of methionine on benzo[a]pyrene (B[a]P)-DNA adducts formation in HepG2 cells. Methionine significantly inhibited B[a]P-DNA adduct formation in HepG2 cells. Methionine significantly decreased the cellular uptake of [(3)H] B[a]P, but increased the cellular discharge of [(3)H] B[a]P from HepG2 cells into the media. B[a]P significantly lowered total cellular glutathione (GSH) level, but co-cultured with B[a]P and methionine, gradually attenuated intracellular GSH levels in a concentration-dependent manner, which was markedly higher at 20-500µM methionine. The cellular proteins of treated cells were resolved by 2D-polyacrylamide gel electrophoresis. Proteomic profiles showed that phase II enzymes such as glutathione S-transferase (GST) omega-1, GSTM3, glyoxalase I (GLO1) and superoxide dismutase (SOD) were down-regulated by B[a]P treatment, whereas cathepsin B (CTSB), Rho GDP-dissociation inhibitor alpha (Rho-GDP-DIA), histamine N-methyltransferase (HNMT), spermidine synthase (SRM) and arginase-1 (ARG1) were up-regulated by B[a]P. B[a]P and methionine treatments, GST omega-1, GSTM3, GLO1 and SOD were significantly enhanced compared to B[a]P alone. Similarly, methionine was effective in diminishing the B[a]P-induced up-regulation of CTSB, Rho-GDP-DIA, HNMT, SRM and ARG1. Our data suggests that methionine might exert a chemoprotective effect on B[a]P-DNA adduct formation by attenuating intracellular GSH levels, blocking the uptake of B[a]P into cells, or by altering expression of proteins involved in DNA adduct formation.


Subject(s)
Benzo(a)pyrene/antagonists & inhibitors , DNA Adducts/antagonists & inhibitors , Liver Neoplasms/chemically induced , Methionine/pharmacology , Proteomics/methods , Arginase/analysis , Cathepsin B/analysis , DNA Adducts/biosynthesis , Glutathione Transferase/analysis , Guanine Nucleotide Dissociation Inhibitors/analysis , Hep G2 Cells , Histamine N-Methyltransferase/analysis , Humans , Lactoylglutathione Lyase/analysis , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Spermidine Synthase/analysis , Superoxide Dismutase/analysis , rho-Specific Guanine Nucleotide Dissociation Inhibitors
5.
Chem Res Toxicol ; 24(6): 877-86, 2011 Jun 20.
Article in English | MEDLINE | ID: mdl-21574630

ABSTRACT

The polyphenolics in green tea are believed to be the bioactive components. However, poor bioavailability following ingestion limits their efficacy in vivo. In this study, polyphenon E (poly E), a standardized green tea extract, was administered by sustained-release polycaprolactone implants (two, 2-cm implants; 20% drug load) grafted subcutaneously or via drinking water (0.8% w/v) to female S/D rats. Animals were treated with continuous low dose of benzo[a]pyrene (BP) via subcutaneous polymeric implants (2 cm; 10% load) and euthanized after 1 and 4 weeks. Analysis of lung DNA by (32)P-postlabeling resulted in a statistically significant reduction (50%; p = 0.023) of BP-induced DNA adducts in the implant group; however, only a modest (34%) but statistically insignificant reduction occurred in the drinking water group at 1 week. The implant delivery system also showed significant reduction (35%; p = 0.044) of the known BP diolepoxide-derived DNA adduct after 4 weeks. Notably, the total dose of poly E administered was >100-fold lower in the implant group than the drinking water group (15.7 versus 1,632 mg, respectively). Analysis of selected phase I, phase II, and nucleotide excision repair enzymes at both mRNA and protein levels showed no significant modulation by poly E, suggesting that the reduction in the BP-induced DNA adducts occurred presumably due to known scavenging of the antidiolepoxide of BP by the poly E catechins. In conclusion, our study demonstrated that sustained systemic delivery of poly E significantly reduced BP-induced DNA adducts in spite of its poor bioavailability following oral administration.


Subject(s)
Benzo(a)pyrene/toxicity , Carcinogens, Environmental/toxicity , Catechin/analogs & derivatives , DNA Adducts/metabolism , Drug Implants , Tea/chemistry , Animals , Benzo(a)pyrene/antagonists & inhibitors , Benzo(a)pyrene/metabolism , Catechin/administration & dosage , Catechin/pharmacology , DNA Adducts/antagonists & inhibitors , DNA Repair/drug effects , Drug Implants/chemistry , Female , Lung/drug effects , Lung/enzymology , Lung/metabolism , Polyesters/chemistry , Rats , Rats, Sprague-Dawley
6.
Cancer Res ; 71(11): 3904-11, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21487034

ABSTRACT

NF-E2-related factor 2 (Nrf2) is a major cytoprotective gene and is a key chemopreventive target against cancer and other diseases. Here we show that Nrf2 faces a dilemma in defense against 4-aminobiphenyl (ABP), a major human bladder carcinogen from tobacco smoke and other environmental sources. Although Nrf2 protected mouse liver against ABP (which is metabolically activated in liver), the bladder level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the predominant ABP-DNA adduct formed in bladder cells and tissues, was markedly higher in Nrf2(+/+) mice than in Nrf2(-/-) mice after ABP exposure. Notably, Nrf2 protected bladder cells against ABP in vitro. Mechanistic investigations showed that the dichotomous effects of Nrf2 could be explained at least partly by upregulation of UDP-glucuronosyltransferase (UGT). Nrf2 promoted conjugation of ABP with glucuronic acid in the liver, increasing urinary excretion of the conjugate. Although glucuronidation of ABP and its metabolites is a detoxification process, these conjugates, which are excreted in urine, are known to be unstable in acidic urine, leading to delivery of the parent compounds to bladder. Hence, although higher liver UGT activity may protect the liver against ABP, it increases bladder exposure to ABP. These findings raise concerns of potential bladder toxicity when Nrf2-activating chemopreventive agents are used in humans exposed to ABP, especially in smokers. We further show that 5,6-dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) significantly inhibits dG-C8-ABP formation in bladder cells and tissues but does not seem to significantly modulate ABP-catalyzing UGT in liver. Thus, CPDT exemplifies a counteracting solution to the dilemma posed by Nrf2.


Subject(s)
Aminobiphenyl Compounds/antagonists & inhibitors , NF-E2-Related Factor 2/metabolism , Urinary Bladder Neoplasms/chemically induced , Urinary Bladder Neoplasms/metabolism , Aminobiphenyl Compounds/pharmacokinetics , Aminobiphenyl Compounds/urine , Animals , Carcinogens/pharmacokinetics , Cell Line, Tumor , Chemoprevention , Cytoprotection , DNA Adducts/antagonists & inhibitors , DNA Adducts/biosynthesis , DNA Damage , Glucuronosyltransferase/metabolism , Humans , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/genetics , Sulfhydryl Compounds/pharmacology , Thiones/pharmacology , Tumor Cells, Cultured , Urinary Bladder/drug effects , Urinary Bladder/metabolism , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/prevention & control
7.
Pol Merkur Lekarski ; 26(152): 156-61, 2009 Feb.
Article in Polish | MEDLINE | ID: mdl-19388525

ABSTRACT

Chemoprevention refers to prevention of tumor formation due to administration of nontoxic synthetic or natural compounds, which can block or weaken the influence of carcinogens on target cells or even reverse previously formed lesions. In esophageal squamous cell carcinoma--which belongs to the group of the most aggressive tumors of digestive system with poor prognosis--an effective prevention would be strongly expected. Chemopreventive properties of many complex diets and pure natural compounds were evaluated until now. Most studies were performed on rats exposed to chemical carcinogens, usually N-nitroso-N-methylbenzylamine (NMBA). The best effects were achieved after administration of diallyl sulfide and phenethyl isothiocyanate. Lyophilized black raspberries, blackberries and strawberries, as well as products obtained from leaves and buds of tea plant, elagic acid and resveratrol were also very effective. Mechanisms of chemopreventive action were associated with, i.e., activity of enzymes involved in carcinogen metabolic activation (mostly cytochrome P450 isoenzymes), inhibition of DNA adducts formation and modulation of gene expression involved in regulation of proliferation, apoptosis and angiogenesis. However, some agents e.g., perillyl alcohol, could enhance development of proliferative lesions in esophageal epithelium.


Subject(s)
Allyl Compounds/pharmacology , Anticarcinogenic Agents/pharmacology , Carcinoma, Squamous Cell/prevention & control , Esophageal Neoplasms/prevention & control , Fruit , Isothiocyanates/pharmacology , Sulfides/pharmacology , Tea , Animals , Apoptosis , Carcinoma, Squamous Cell/diet therapy , Chemoprevention , DNA Adducts/antagonists & inhibitors , Dimethylnitrosamine/analogs & derivatives , Esophageal Neoplasms/chemically induced , Esophageal Neoplasms/diet therapy , Plant Oils/pharmacology , Rats
8.
Toxicol Appl Pharmacol ; 235(3): 296-304, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19146867

ABSTRACT

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B(1) (AFB(1)) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB(1) treatment. Competitive enzyme immunoassays indicated that after AFB(1) treatment, the AFB(1)-DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB(1)-DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB(1) in the cultural supernatants of cells exposed to AFB(1) showed that the AFM(1) level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB(1). Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB(1). Automated docking experiments showed that AFB(1) binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT-AFB(1) interaction is 44.9 microM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB(1), resulting in reduced AFB(1)-DNA adducts formation and cell death.


Subject(s)
Aflatoxin B1/antagonists & inhibitors , Aflatoxin B1/metabolism , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/prevention & control , Glycine N-Methyltransferase/physiology , Liver Neoplasms, Experimental/enzymology , Liver Neoplasms, Experimental/prevention & control , Aflatoxin B1/toxicity , Animals , Carcinoma, Hepatocellular/chemically induced , Cell Line, Tumor , DNA Adducts/antagonists & inhibitors , DNA Adducts/biosynthesis , Female , Glycine N-Methyltransferase/administration & dosage , Glycine N-Methyltransferase/biosynthesis , Glycine N-Methyltransferase/genetics , Humans , Liver Neoplasms, Experimental/chemically induced , Male , Mice , Mice, Transgenic
9.
Nutr Cancer ; 58(2): 205-12, 2007.
Article in English | MEDLINE | ID: mdl-17640167

ABSTRACT

Recent studies from our laboratory have shown that the transactivation of nuclear factor kappa B (NF kappa B) and activator protein-1 (AP-1) plays an important mechanistic role in ultraviolet (UV)-induced skin carcinogenesis in mice. We also demonstrated that a methanol extract (ME) fraction from black raspberries (Rubus occidentalis) (RO; RO-ME) inhibits benzo[a]pyrene-7,8-diol-9,10-epoxide [B(a)PDE]-induced activation of NF kappa B and AP-1 in cultured mouse epidermal cells. In the present study, we determined if RO-ME might also inhibit the induction of NF kappa B and AP-1 in mouse epidermal cells exposed to mid UV radiation (UVB) and short UV radiation (UVC) and whether methanol fractions from strawberries and blueberries would also be effective. Our results showed that RO-ME inhibited UVB-induced activation of NF kappa B in mouse epidermal cells in a time- and dose-dependent manner; however, the methanol fractions from strawberries and blueberries were ineffective. Interestingly, none of the fractions from all 3 berry types inhibited UVB- or UVC-induced activation of AP-1, suggesting that inhibition of UV-induced signaling pathways is specific for black raspberries and NF kappa B. Cyanidin-3-rutinoside, an anthocyanin found in abundance in black raspberries and not in strawberries or high-bush blueberries, was found to contribute to the inhibition of UVB-induced activation of NF kappa B. These results suggest that berries differ in their ability to influence signaling pathways leading to activation of NF kappa B and AP-1 when using UV light as the inducer.


Subject(s)
Fruit/chemistry , NF-kappa B/antagonists & inhibitors , Plant Extracts/pharmacology , Transcription Factor AP-1/antagonists & inhibitors , 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide/toxicity , Animals , Blueberry Plants/chemistry , Carcinogens/antagonists & inhibitors , Carcinogens/toxicity , Cell Line , DNA Adducts/antagonists & inhibitors , DNA Adducts/toxicity , Dose-Response Relationship, Drug , Fragaria/chemistry , Mice , NF-kappa B/drug effects , NF-kappa B/radiation effects , Species Specificity , Time Factors , Transcription Factor AP-1/drug effects , Transcription Factor AP-1/radiation effects
10.
J Inorg Biochem ; 101(8): 1153-64, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17592741

ABSTRACT

The presence of polyamines in living cells is crucial for survival. Due to their high net charge at physiological pH, polyamines effectively charge neutralize the phosphodiester backbone of DNA in an interaction that also may protect the DNA from external damage. We here present a study illustrating the influence of spermidine and spermine on the platination reactions of the model oligonucleotides d(T(6)GT(6)), d(T(12)GT(12)), and d(T(24)GT(24)), and the pUC18 DNA plasmid. The aquated forms of the anticancer active compounds cisplatin (cis-[Pt(NH(3))(2)Cl(2)]) and the major Pt(II) metabolite of JM216 (cis-[PtCl(2)(NH(3))(c-C(6)H(11)NH(2))], JM118) were used as platination reagents. The study shows that the kinetics for formation of the coordinative Pt-DNA adduct are strongly influenced by the presence of sub-millimolar polyamine concentrations. At polyamine concentrations in the muM-range, the reactions remain salt-dependent. In contrast, platination of pUC18 is effectively prevented at mM concentrations of both spermidine and spermine with the latter as the more potent inhibitor. The results suggest that variations of intracellular polyamine concentrations may have a profound influence on the efficacy by which cationically charged reagents interfere with DNA function in vivo by modulation of the preassociation conditions.


Subject(s)
DNA Adducts/antagonists & inhibitors , DNA, Single-Stranded/antagonists & inhibitors , Oligonucleotides/antagonists & inhibitors , Platinum Compounds/antagonists & inhibitors , Spermidine/pharmacology , Spermine/pharmacology , Cations, Monovalent , DNA Adducts/metabolism , DNA, Single-Stranded/metabolism , Dose-Response Relationship, Drug , Kinetics , Oligonucleotides/metabolism , Plasmids/antagonists & inhibitors , Plasmids/metabolism , Platinum Compounds/metabolism , Sodium/chemistry , Sodium/metabolism
11.
Free Radic Biol Med ; 42(8): 1258-69, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17382206

ABSTRACT

4-hydroxynonenal (HNE), one of the main breakdown products of lipid peroxides, has been shown to react with DNA yielding a 1,N2-propano adduct to 2'-deoxyguanosine. However, HNE may also react with a wide range of biomolecules before reaching the nucleus. Glutathione (GSH), the most abundant cellular thiol-containing peptide, is likely to be a major cytosolic target for HNE because of its high reactivity and its implication in the detoxification of this aldehyde. In order to estimate the proportion of HNE actually reaching DNA in human THP1 monocytes, we designed an experimental protocol aimed at quantifying DNA adducts and HNE-GSH in the same sample of cells exposed to extracellularly added HNE. Reverse-phase HPLC associated with tandem mass spectrometry detection was used as the analytical tool. It was first observed that, once produced, the HNE-GSH conjugate was very efficiently excreted from the cells into the culture medium. More strikingly, we determined that the amount of HNE-GSH conjugate produced was 4 orders of magnitude higher than that of DNA adduct. These results emphasize the major role played by glutathione in the protection of DNA against electrophilic species.


Subject(s)
Aldehydes/pharmacokinetics , DNA Adducts/antagonists & inhibitors , Glutathione/metabolism , Aldehydes/chemistry , Animals , Cell Line , Cell Survival , DNA Adducts/chemistry , Deoxyguanosine/chemistry , Glutathione/chemistry , Humans , Models, Biological , Models, Molecular , Monocytes/physiology , Solutions
12.
Nutr Cancer ; 54(1): 33-46, 2006.
Article in English | MEDLINE | ID: mdl-16800771

ABSTRACT

For several years, our laboratory has been evaluating the ability of lyophilized (freeze-dried) black raspberries (Rubus occidentalis, BRBs), blackberries (R. fructicosus, BBs), and strawberries (Fragaria ananasia, STRWs) to inhibit carcinogen-induced cancer in the rodent esophagus. To assure "standardized" berry preparations for study, each berry type is of the same cultivar, picked at about the same degree of ripeness, washed and frozen within 2-4 h of the time of picking, and freeze-dried under conditions that preserve the components in the berries. Some of the known chemopreventive agents in berries include vitamins A, C, and E and folic acid; calcium and selenium; beta-carotene, alpha-carotene, and lutein; polyphenols such as ellagic acid, ferulic acid, p-coumaric acid, quercetin, and several anthocyanins; and phytosterols such as beta-sitosterol, stigmasterol, and kaempferol. In initial bioassays, freeze-dried STRW, BRB, and BB powders were mixed into AIN-76A synthetic diet at concentrations of 5% and 10% and fed to Fischer 344 rats before, during, and after treatment with the esophageal carcinogen N-nitrosomethylbenzylamine (NMBA). At 25 wk of the bioassay, all three berry types were found to inhibit the number of esophageal tumors (papillomas) in NMBA-treated animals by 24-56% relative to NMBA controls. This inhibition correlated with reductions in the formation of the NMBA-induced O6-methylguanine adduct in esophageal DNA, suggesting that the berries influenced the metabolism of NMBA leading to reduced DNA damage. Studies are ongoing to determine the mechanisms by which berries influence NMBA metabolism and DNA adduct formation. BRBs and STRWs were also tested in a postinitiation scheme and were found to inhibit NMBA-induced esophageal tumorigenesis by 31-64% when administered in the diet following treatment of the animals with NMBA. Berries, therefore, inhibit tumor promotion and progression events as well as tumor initiation. In vivo mechanistic studies with BRBs indicate that they reduce the growth rate of premalignant esophageal cells, in part, through down-regulation of cyclooxygenase-2 leading to reduced prostaglandin production and of inducible nitric oxide synthase leading to reduced nitrate/nitrite levels in the esophagus. Based upon the preclinical data on rodents, we have initiated prevention trials in humans to determine if berries might exhibit chemopreventive effects in the esophagus.


Subject(s)
Diet , Esophageal Neoplasms/prevention & control , Fruit , Animals , Anticarcinogenic Agents/administration & dosage , Anticarcinogenic Agents/analysis , Cyclooxygenase 2/genetics , DNA Adducts/antagonists & inhibitors , Dimethylnitrosamine/analogs & derivatives , Dinoprostone/analysis , Esophageal Neoplasms/chemically induced , Esophagus/chemistry , Food Preservation , Fragaria/chemistry , Freeze Drying , Fruit/chemistry , Gene Expression/drug effects , Guanosine/analogs & derivatives , Nitric Oxide Synthase Type II/genetics , Nitrites/analysis , Phytotherapy , Proliferating Cell Nuclear Antigen/analysis , Rats , Rats, Inbred F344 , Rosaceae/chemistry , Weight Gain
13.
Int J Oncol ; 28(5): 1131-40, 2006 May.
Article in English | MEDLINE | ID: mdl-16596229

ABSTRACT

There is evidence that aspirin and other non-steroidal anti-inflammatory drugs may be protective agents against cancer in the gastrointestinal tract. These effects are particularly well documented for the colon and rectum. Some epidemiological and experimental studies have suggested that aspirin could also be a chemopreventive agent against breast cancer. We investigated the effects of the aspirin metabolite, salicylate (SA), on 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adduct formation as well as on the expression of the enzymes involved in the carcinogen bioactivation pathway, in particular cytochrome P450 1A (CYP1A) and cyclooxygenases (COX-1 and COX-2). The effects of the test drug were examined in both the human mammary carcinoma cell line, MCF-7, and mammary cells derived from DMBA-induced rat mammary tumours (RMTCs). In this study, we also reported the effects of SA on cell growth and viability in breast cancer cells (BCCs). The results demonstrated that DMBA-DNA adduct formation in both cancer cell lines was inhibited by SA at concentrations of > or = 2.5 mM. CYP1A was undetectable in RMTCs while CYP1A induction by beta-naphthoflavone in MCF-7 cells was significantly inhibited by SA in a concentration-dependent manner. Aspirin did not affect COX-1 expression in either of the BCCs. COX-2 was not detected in MCF-7 cells, but its expression in RMTCs was inhibited by SA treatment, which also significantly reduced BCC growth, but failed to cause cell death by necrosis or apoptosis. These data suggest that inhibition of DMBA-DNA adduct formation may contribute to aspirin breast cancer chemopreventive action and indicate that this drug can act in the first stage of carcinogenesis.


Subject(s)
9,10-Dimethyl-1,2-benzanthracene/analogs & derivatives , DNA Adducts/antagonists & inhibitors , Salicylates/pharmacology , 9,10-Dimethyl-1,2-benzanthracene/antagonists & inhibitors , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cyclooxygenase 1/genetics , Cyclooxygenase 2/genetics , Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Cytochrome P-450 CYP1A1/genetics , Female , Humans , Mammary Neoplasms, Experimental/pathology , Rats , Rats, Sprague-Dawley
14.
Toxicol Appl Pharmacol ; 213(2): 98-104, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16307765

ABSTRACT

Perillyl alcohol (POH) is a dietary monoterpene with potential applications in chemoprevention and chemotherapy. Although clinical trials are under way, POH's physiological and pharmacological properties are still unclear. In the present study, the effect of POH on polycyclic aromatic hydrocarbon (PAH)-induced genotoxicity, and the related expression were examined in MCF-7 cells. Exposure to environmental toxicant increases the risk of cancer. Many of these compounds are pro-carcinogens and are biotransformed into their ultimate genotoxic structures by xenobiotic metabolizing enzymes. CYP1A1 and 1B1 are enzymes that catalyze the biotransformation of dimethylbenz[a]anthracene (DMBA). Our data revealed that 0.5 microM of POH was effective in blocking DMBA-DNA binding. Ethoxyresorufin-O-deethylase (EROD) assay indicated that the administration of POH inhibited the DMBA-induced enzyme activity in MCF-7 cells. Enzyme kinetic analysis revealed that POH inhibited CYP1B1 but not CYP1A1 activity. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay also demonstrated that the monoterpene reduced CYP1B1 mRNA abundance induced by DMBA. The present study illustrated that POH might inhibit and downregulate CYP1B1, which could protect against PAH-induced carcinogenesis.


Subject(s)
Aryl Hydrocarbon Hydroxylases/drug effects , Cytochrome P-450 CYP1A1/drug effects , DNA Adducts/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Epithelial Cells/enzymology , Monoterpenes/pharmacology , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Antineoplastic Agents/pharmacology , Aryl Hydrocarbon Hydroxylases/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Carcinogens/toxicity , Cell Line, Tumor , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1B1 , DNA Damage/physiology , Down-Regulation , Drug Interactions , Epithelial Cells/drug effects , Female , Humans , Inhibitory Concentration 50 , Polycyclic Aromatic Hydrocarbons/toxicity , Transcriptional Activation/drug effects , Xenobiotics/toxicity
15.
Biochemistry ; 44(18): 7059-68, 2005 May 10.
Article in English | MEDLINE | ID: mdl-15865450

ABSTRACT

The importance of DNA polymerase-DNA minor groove interactions on translesion synthesis (TLS) was examined in vitro using variants of exonuclease-deficient Klenow polymerase and site-specifically modified DNA oligonucleotides. Polymerase variant R668A lacks primer strand interactions, while variant Q849A lacks template strand interactions. O(6)-Methylguanine (m6G) and abasic site TLS was examined in three stages: dNTP insertion opposite the lesion, extension from a terminal lesion-containing base pair, and the dissociation equilibrium of the polymerase from the lesion-containing template. Less than 5% TLS was observed at the insertion step for either variant on the lesion-containing templates. While extensive TLS was observed for WT polymerase on the m6G template, only incorporation opposite the lesion was observed for the R668A variant. Loss of the template strand interaction, Q849A, resulted in the inability to insert dNTPs opposite either the m6G or abasic lesion. For both variants, extension of purine-containing m6G primer-templates was increased relative to WT polymerase. We observed similar extension efficiencies for all variants, relative to WT, using abasic template-primers. Polymerase dissociation/reassociation was studied through the use of a competitor primer/template complex. Dissociation for WT polymerase increased 2-fold and 3-fold, respectively, for m6G and abasic lesion-containing templates, relative to the natural template. Variants lacking DNA minor groove interactions displayed increased dissociation from DNA templates, relative to WT polymerase, but do not display an increased level of lesion-induced polymerase dissociation. Our results indicate that the primer and template strand interactions of the Klenow polymerase with the DNA minor groove are critical for maintaining the DNA-polymerase complex during translesion synthesis.


Subject(s)
DNA Damage , DNA Polymerase I/genetics , DNA Polymerase I/metabolism , DNA Repair , DNA, Bacterial/metabolism , Exonucleases/deficiency , Guanine/analogs & derivatives , Guanine/antagonists & inhibitors , Nucleic Acid Conformation , Amino Acid Substitution/genetics , Base Pair Mismatch/genetics , DNA Adducts/antagonists & inhibitors , DNA Adducts/biosynthesis , DNA Polymerase I/chemistry , DNA Primers/metabolism , DNA, Bacterial/biosynthesis , DNA, Bacterial/chemistry , Exonucleases/metabolism , Guanine/biosynthesis , Oligodeoxyribonucleotides/biosynthesis , Oligodeoxyribonucleotides/metabolism , Substrate Specificity/genetics , Templates, Genetic
16.
J Agric Food Chem ; 53(3): 812-5, 2005 Feb 09.
Article in English | MEDLINE | ID: mdl-15686438

ABSTRACT

An evaluation of the in vivo antigenotoxic potential of beer components on heterocyclic amines including 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 3-amino-1-methyl-5H-pyrido[4.3-b]indole (Trp-P-2) was determined with particular focus on the target organs of tumorigenesis, and the protective mechanisms involved were investigated. Beer-solution, consisting of a freeze-dried and dissolved sample, given as drinking water, reduced the formation of MeIQx-DNA adducts in mouse liver and lungs. Beer-solution added in the diet as a mimic of food additives also significantly reduced the amount of DNA adducts present in the liver, lung, and kidney DNA of mice fed with MeIQx compared to control mice fed with MeIQx in the absence of beer-solution. The amount of adducts present in the liver of mice with single or continuous administration of Trp-P-2 was significantly reduced when beer-solution was given as part of the diet compared to control mice given Trp-P-2 without beer-solution. Protective effects were observed both with lager- and stout-type samples. In an effort to investigate the mechanisms responsible for the observed protective effects, the effects of beer-solution on metabolizing enzymes for heterocyclic amines were examined. Beer-solutions inhibited the metabolic activation of Trp-P-2 to Trp-P-2(NHOH), as demonstrated by HPLC analysis. Considering the overall suppression of the genotoxicity of MeIQx and Trp-P-2 by beer, we have shown that beer components can inhibit the metabolic activation of heterocyclic amines and subsequently suppress the observed genotoxicity. The results of this study show that beer components are protective against the genotoxic effects of heterocyclic amines on target organs associated with tumorigenesis in vivo.


Subject(s)
Beer , Carbolines/pharmacology , DNA Adducts/antagonists & inhibitors , Liver/chemistry , Lung/chemistry , Quinoxalines/pharmacology , Animals , Male , Mice , Mice, Inbred C57BL , Mutagens/pharmacology
17.
Anticancer Drugs ; 16(2): 129-36, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15655409

ABSTRACT

Raf kinase plays a central role in oncogenic signaling and acts as a downstream effector of Ras in the extracellular signal-regulated (ERK) kinase pathway. BAY 43-9006 (BAY) is a novel signal transduction inhibitor that prevents tumor cell proliferation and angiogenesis through blockade of the Raf/MEK/ERK pathway at the level of Raf kinase and the receptor tyrosine kinases vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-beta. The present study evaluates the effects of combining BAY and platinum derivatives on human colorectal cancer cells using different incubation protocols. Our data show that the combination of oxaliplatin or cisplatin with BAY results in marked antagonism irrespective of the used application schedule. Furthermore, BAY abrogates the cisplatin-induced G2 arrest as well as the G1 arrest induced by oxaliplatin. BAY alone arrests cancer cells in their current cell cycle phase and affects cell cycle regulative genes. Specifically, BAY reduced the protein expression of p21Cip1 as well as cyclin D1, and inhibits the expression of cdc2 (cdk1). Utilizing atom absorption spectrometry, BAY significantly reduced cellular uptake of platinum compounds and thereby the generation of DNA adducts. Taken together, co-incubation with BAY results in reduced cellular uptake of platinum compounds and consecutively reduced generation of DNA adducts, and eventually decreased cellular cytotoxicity in human colorectal cancer cells. Our results indicate that the Raf kinase inhibitor BAY 43-9006 might also directly or indirectly interact with platinum transporter proteins in vitro.


Subject(s)
Antineoplastic Agents/pharmacology , Benzenesulfonates/pharmacology , Cisplatin/pharmacology , Organoplatinum Compounds/pharmacology , Pyridines/pharmacology , raf Kinases/antagonists & inhibitors , Antineoplastic Agents/pharmacokinetics , Blotting, Western , Cell Cycle/drug effects , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Cisplatin/pharmacokinetics , Cyclin D1/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p21 , DNA Adducts/antagonists & inhibitors , Drug Antagonism , Humans , Niacinamide/analogs & derivatives , Organoplatinum Compounds/pharmacokinetics , Phenylurea Compounds , Pyridines/pharmacokinetics , Sorafenib
18.
Cancer Res ; 64(18): 6797-804, 2004 Sep 15.
Article in English | MEDLINE | ID: mdl-15374999

ABSTRACT

The mechanisms by which n-3 polyunsaturated fatty acids (PUFAs) decrease colon tumor formation have not been fully elucidated. Examination of genes up- or down-regulated at various stages of tumor development via the monitoring of gene expression relationships will help to determine the biological processes ultimately responsible for the protective effects of n-3 PUFA. Therefore, using a 3 x 2 x 2 factorial design, we used Codelink DNA microarrays containing approximately 9000 genes to help decipher the global changes in colonocyte gene expression profiles in carcinogen-injected Sprague Dawley rats. Animals were assigned to three dietary treatments differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-3 PUFA, or olive oil/n-9 monounsaturated fatty acid), two treatments (injection with the carcinogen azoxymethane or with saline), and two time points (12 hours and 10 weeks after first injection). Only the consumption of n-3 PUFA exerted a protective effect at the initiation (DNA adduct formation) and promotional (aberrant crypt foci) stages. Importantly, microarray analysis of colonocyte gene expression profiles discerned fundamental differences among animals treated with n-3 PUFA at both the 12 hours and 10-week time points. Thus, in addition to demonstrating that dietary fat composition alters the molecular portrait of gene expression profiles in the colonic epithelium at both the initiation and promotional stages of tumor development, these findings indicate that the chemopreventive effect of fish oil is due to the direct action of n-3 PUFA and not to a reduction in the content of n-6 PUFA.


Subject(s)
Anticarcinogenic Agents/pharmacology , Colonic Neoplasms/genetics , Colonic Neoplasms/prevention & control , Fatty Acids, Unsaturated/pharmacology , Triglycerides/pharmacology , Animals , Apoptosis/drug effects , Azoxymethane , Carcinogens , Cell Differentiation/drug effects , Colonic Neoplasms/metabolism , DNA Adducts/antagonists & inhibitors , DNA Adducts/biosynthesis , Disease Progression , Down-Regulation/drug effects , Eating/drug effects , Fatty Acids, Omega-3 , Fatty Acids, Omega-6/pharmacology , Fish Oils/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Male , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Rats , Rats, Sprague-Dawley , Up-Regulation/drug effects , Weight Gain/drug effects
19.
Mutat Res ; 551(1-2): 181-97, 2004 Jul 13.
Article in English | MEDLINE | ID: mdl-15225592

ABSTRACT

Searching for optimal diets and for naturally occurring agents in routinely consumed foods that may inhibit cancer development, although challenging, constitutes a valuable and plausible approach to finding ways to control and prevent cancer. To date, the use of the micronutrient selenium in human clinical trials is limited but the outcome of these investigations indicates that selenium is one of the most promising agents. Data presented in this mini-review indicate that the dose and the form (structure) in which selenium is used are the most critical determinants of success in future clinical trials. The focus of this mini-review is on the mechanisms of mammary cancer chemoprevention by organoselenium compounds. Among the naturally occurring organoselenium compounds, Se-Methylselenocysteine is more efficacious than the most extensively studied forms, such as selenomethionine. However, we showed that synthetic organoselenium compounds can be tailored to achieve greater chemopreventive efficacy with minimal side effects by structural modifications; it is evident that synthetic agents are superior to the inorganic selenite, naturally occurring selenium compounds and their sulfur-containing analogs. We have demonstrated that 1,4-phenylenebis (methylene) selenocyanate (p-XSC) and its putative metabolite glutathione conjugate (p-XSeSG) are highly promising agents in the chemoprevention of mammary carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)-rat mammary tumor model system. Both compounds inhibit the initiation phase of carcinogenesis by inhibiting DMBA-DNA adduct formation in the target organ in vivo. cDNA microarray analysis indicates that both selenium compounds alter genes in a manner that leads to inhibition of cell proliferation and induction of apoptosis; modulation of apoptosis and cell proliferation can account for chemoprevention during the post-initiation phase of mammary carcinogenesis. Using a rat mammary cancer cell line, we compared p-XSC and p-XSeSG as inhibitors of cell proliferation; depending on the selenium dose and time point selected, p-XSC was comparable to or better than p-XSeSG. Collectively, the results described here, suggest that the molecular targets modulated by organoselenium compounds are highly useful indicators of success in clinical cancer chemoprevention trials.


Subject(s)
9,10-Dimethyl-1,2-benzanthracene/analogs & derivatives , Breast Neoplasms/prevention & control , Organoselenium Compounds/pharmacology , 9,10-Dimethyl-1,2-benzanthracene/antagonists & inhibitors , Animals , Anticarcinogenic Agents , Clinical Trials as Topic , DNA Adducts/antagonists & inhibitors , Humans , Mammary Neoplasms, Experimental/prevention & control , Organoselenium Compounds/metabolism
20.
In Vivo ; 18(1): 21-31, 2004.
Article in English | MEDLINE | ID: mdl-15011747

ABSTRACT

Shikonin has the potential to prevent, or be used in the treatment of, bladder transitional cell carcinoma induced by arylamines. We evaluated its effectiveness by measuring the amount of acetylated 2-aminofluorene (AF), AF-DNA adducts, changes of NAT mRNA and the amount of NAT enzyme. T24 human bladder cancer cells were incubated with 30 microM AF with different concentrations of shikonin for various times. T24 cells treated with shikonin (16 microM) were then harvested and used in 2 experiments: 1). T24 cells were incubated with 22.5 microM AF and shikonin (0, 16 microM) (co-treatment) for 6, 12, 18, 24 and 48 h. 2). T24 cells were incubated with various concentrations of AF and shikonin (0, 16 microM) for 24 h. AF and AAF were measured by HPLC. Then in the prepared human T24 cell cytosols different concentrations of AF and shikonin were added to measure the kinetic constants of NAT. Next, AF-DNA adducts in human T24 cells with or without treatment with shikonin were detected and measured. The final two steps included measuring the NAT Ag-Ab complex after treatment with and without shikonin and evaluating the effect of shikonin on the NAT genes. Higher concentrations of shikonin induced decreasing AF acetylation. We found that the longer the culture period, the greater the difference in AF acetylation in the same shikonin concentrations. It was also noted that increase in AAF was proportional to incubation time. In the presence of 16 microM of shikonin, N-acetylation of AF decreased by up to 72-84%. Shikonin decreased the amount of AAF production in human T24 cells in all examined AF doses. Both Km and Vmax values in the cytosolic NAT decreased after the addition of shikonin to the cytosol. Finally, shikonin decreased the amount of AAF production and AF-DNA adducts formation in human 724 cells in all examined AF doses. The percentage of cells stained by antibody was significantly different after treatment with shikonin, especially with the higher shikonin concentrations. The NAT1 mRNA level and the NAT1/beta-actin ratio decreased significantly with higher concentrations (16-24 microM) of shikonin. Shikonin affected NAT activity, gene expression (NAT1 mRNA), AF-DNA adducts formation and formation of NAT Ag-Ab in human bladder tumor T24 cells. Therefore, shikonin should be considered as a candidate agent for the prevention or treatment of transitional cell carcinoma.


Subject(s)
Arylamine N-Acetyltransferase/antagonists & inhibitors , Arylamine N-Acetyltransferase/genetics , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Naphthoquinones/pharmacology , Urinary Bladder Neoplasms/enzymology , Aged , Aged, 80 and over , Animals , Arylamine N-Acetyltransferase/biosynthesis , Cell Line, Tumor , DNA Adducts/antagonists & inhibitors , DNA Adducts/metabolism , Dose-Response Relationship, Drug , Female , Humans , Mice , Mice, Inbred BALB C , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics
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