Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Methoxymorpholinyl doxorubicin (MMDX) is a novel anti-cancer anthracycline that differs from doxorubicin in its mechanisms of action, pattern of resistance and metabolism. Whereas doxorubicin is primarily an inhibitor of topoisomerase II, MMDX inhibits both topoisomerases I and II, resulting in predominantly single-strand DNA cleavage and, to a lesser extent, double-strand DNA breakage. MMDX is equally cytotoxic in vitro against the doxorubicin-sensitive and -resistant uterine sarcoma cell lines, MES-SA and Dx5. Using fluorescent laser cytometry, MMDX was retained intracellularly to a similar extent in MES-SA and Dx5; the intracellular retention of MMDX was 7.5-fold higher than that of doxorubicin in Dx5. The cytotoxicity of MMDX on an ovarian carcinoma cell line, ES-2, was potentiated 50-fold by preincubating the drug with human liver microsomes and NADPH. This cytotoxic potentiation was associated with the appearance of DNA interstrand cross-links. The in vitro potentiation of MMDX was inhibited by cyclosporin A, which is a substrate for human cytochrome P450 IIIA.
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PMID:Metabolic conversion of methoxymorpholinyl doxorubicin: from a DNA strand breaker to a DNA cross-linker. 801 45

NAD(P)H dependent cytochrome P450's and other haemoproteins under hypoxia, mediate two-electron reduction of a wide range of structurally dissimilar N-oxides to their respective tertiary amines. Metabolic reduction can be utilised, in acute and chronic hypoxia, to convert N-oxides of DNA affinic agents to potent and persistent cytotoxins. In this respect a knowledge of N-oxide bioreduction and the importance of the cationic nature of agents that bind to DNA by intercalation can be combined to rationalise N-oxides as prodrugs of DNA binding agents. The concept is illustrated using the alkylaminoanthraquinones which are a group of cytotoxic agents with DNA binding affinity that is dependent on the cationic nature of these compounds. The actions of the alkylaminoanthraquinones involve drug intercalation into DNA (and double stranded RNA) and inhibition of both DNA and RNA polymerases and topoisomerase Type I and II. A di-N-oxide analogue of mitoxantrone, 1,4-bis([2-(dimethylamino-N-oxide)ethyl]amino)5,8-dihydroxyanthracene -9,10- dione (AQ4N) has been shown to possess no intrinsic binding affinity for DNA and has low toxicity. Yet in the absence of air AQ4N can be reduced in vitro to a DNA affinic agent with up to 1000-fold increase in cytotoxic potency. Importantly the reduction product, AQ4, is stable under oxic conditions. Studies in vivo indicate that antitumour activity of AQ4N is manifest under conditions that promote transient hypoxia and/or diminish the oxic tumour fraction. The advantage of utilising the reductive environment of hypoxic tumours to reduce N-oxides is that, unlike conventional bioreductive agents, the resulting products will remain active even if the hypoxia that led to bioactivation is transient or the active compounds, once formed, diffuse away from the hypoxic tumour regions. Furthermore, the DNA affinic nature of the active compounds should ensure their localisation in tumour tissue.
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PMID:Rationale for the use of aliphatic N-oxides of cytotoxic anthraquinones as prodrug DNA binding agents: a new class of bioreductive agent. 837 16

Cytochrome P450 (P450) enzyme expression patterns were determined for a panel of 60 human tumor cell lines, representing nine tumor tissue types, used by the National Cancer Institute (NCI) Anticancer Drug Screening Program. All 60 tumor cell lines displayed significant P450 activity, as well as P450 reductase activity, as determined using the general P450 substrate 7-benzyloxyresorufin. Cell line-specific P450 enzyme patterns were observed using three other P450 substrates, 7-ethoxycoumarin, coumarin, and 7-ethoxyresorufin, each of which was metabolized at a low rate. Using a pattern-matching computer program, COMPARE, correlative relationships were investigated between the arrays of P450 activities and the patterns of cytotoxicity exhibited by a large group of anticancer agents of proven or potential clinical utility. Significant negative correlations between the patterns of P450-dependent 7-benzyloxyresorufin metabolism activity and cell line chemosensitivity were observed for 10 standard anticancer agents (including 6 alkylating agents) and 55 investigational compounds, suggesting a role for P450 metabolism in the inactivation of these agents. Negative correlations between 7-ethoxycoumarin O-deethylation and cell line chemosensitivity to a group of topoisomerase inhibitors were also seen, again suggesting P450-dependent drug inactivation. P450 enzyme profiling may thus aid in interpreting the patterns of drug sensitivity and resistance in the NCI tumor cell panel, and may facilitate the identification of anticancer agents whose activity can be altered via cytochrome P450 metabolism.
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PMID:P450 enzyme expression patterns in the NCI human tumor cell line panel. 1118

St. John's wort (Hypericum perforatum) is the most widely used herbal medicine for the treatment of depression. However, concerns have arisen about the potential of its interaction with other drugs due to the induction of cytochrome P450 isozymes 1A2 and 3A4 by the components hypericin and hyperforin, respectively. Structurally similar natural products are often employed as antitumor agents due to their action as inhibitors of DNA topoisomerases, nuclear enzymes that modify DNA during cellular proliferation. Preliminary findings that hypericin inhibited the DNA relaxation activity of topoisomerase IIalpha (topo II; EC 5.99.1.3) led us to investigate the mechanism of enzyme inhibition. Rather than stabilizing the enzyme in covalent complexes with DNA (cleavage complexes), hypericin inhibited the enzyme prior to DNA cleavage. In vitro assays indicate that hypericin is a potent antagonist of cleavage complex stabilization by the chemotherapeutics etoposide and amsacrine. This antagonism appears to be due to the ability of hypericin to intercalate or distort DNA structure, thereby precluding topo II binding and/or DNA cleavage. Supporting its non-DNA damaging, catalytic inhibition of topo II, hypericin was shown to be equitoxic to both wild-type and amsacrine-resistant HL-60 leukemia cell lines. Moreover, hypericin was incapable of stimulating DNA damage-responsive gene promoters that are activated by etoposide. As with the in vitro topo II assay, antagonism of DNA damage stimulated by 30 microM etoposide was evident in leukemia cells pretreated with 5 microM hypericin. Since many cancer patients experience clinical depression and concomitantly self-medicate with herbal remedies, extracts of St. John's wort should be investigated further for their potential to antagonize topo II-directed chemotherapy regimens.
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PMID:Catalytic inhibition of human DNA topoisomerase IIalpha by hypericin, a naphthodianthrone from St. John's wort (Hypericum perforatum). 1159 74

Ellipticine is a potent antitumor agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Using [3H]-labeled ellipticine, we observed substantial microsome (cytochrome P450)-dependent binding of ellipticine to DNA. In rat, rabbit, minipig, and human microsomes, in reconstituted systems with isolated cytochromes P450 and in Supersomes containing recombinantly expressed human cytochromes P450, we could show that ellipticine forms a covalent DNA adduct detected by [32P]-postlabeling. The most potent human enzyme is CYP3A4, followed by CYP1A1, CYP1A2, CYP1B1, and CYP2C9. Another minor adduct is formed independent of enzymatic activation. The [32P]-postlabeling analysis of DNA modified by activated ellipticine confirms the covalent binding to DNA as an important type of DNA modification. The DNA adduct formation we describe is a novel mechanism for the ellipticine action and might in part explain its tumor specificity.
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PMID:The anticancer agent ellipticine on activation by cytochrome P450 forms covalent DNA adducts. 1175 21

Ellipticine is a potent antineoplastic agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). We examined a panel of genetically engineered V79 cell lines including the parental line V79MZ and recombinant cells expressing the human CYP enzymes CYP1A1, CYP1A2 or CYP3A4 for their ability to activate ellipticine. The extent of activation was determined by analysing DNA adducts by 32P-postlabelling. Ellipticine was found to be toxic to all V79 cell lines with IC(50) values ranging from 0.25 to 0.40 microM. The nuclease P1 version of the 32P-postlabelling assay yielded a similar pattern of ellipticine-DNA adducts with two major adducts in all cells, the formation of only one of which was dependent on CYP activity. This pattern is identical to that detected in DNA reacted with ellipticine and the reconstituted CYP enzyme system in vitro as confirmed by HPLC of the isolated adducts. Total adduct levels ranged from 2 to 337 adducts per 10(8) nucleotides, in the parental line and in V79 expressing CYP3A4, respectively. As in vitro, human CYP1A2 and CYP1A1 were less active. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in cells in culture, and confirm the formation of covalent DNA adducts as a new mechanism of ellipticine action.
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PMID:Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes. 1212 50

Aliphatic amine N-oxides have long been identified as non-toxic metabolites of a large number of tertiary amines drugs. Bioreduction of such N-oxides will generate the active parent amine. This principle has been adopted to develop AQ4N, a di-N-oxide anticancer prodrug with little intrinsic cytotoxicity. However, AQ4N is bioreduced in hypoxic regions of solid tumors and micrometastatic deposits to generate a cytotoxic alkylaminoanthraquinone metabolite. The 4-electron reduction metabolite of AQ4N has high affinity for DNA and is a potent inhibitor of topoisomerase II, a DNA processing enzyme crucial to cell division. The development of AQ4N has proceeded on many fronts in order to establish this unique anticancer prodrug opportunity. Preclinical studies in vivo have demonstrated that although AQ4N has little or no intrinsic cytotoxic activity per se it (i) enhances the antitumor effects of radiation and conventional chemotherapeutic agents, (ii) is pharmacokinetically stable, and (iii) is a substrate for cytochrome P450 (CYP). A study of AQ4N metabolism in vitro and ex vivo using purified CYP enzymes, phenotyped human livers and CYP transfected cell lines shows that CYP3A, 1A and 1B1 family members contribute to AQ4N bioreduction in the absence of oxygen. Importantly AQ4N is shown to be metabolized by tumors known to express CYP isoforms. AQ4N is currently in Phase I clinical trials.
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PMID:Bioreductively activated antitumor N-oxides: the case of AQ4N, a unique approach to hypoxia-activated cancer chemotherapy. 1221 68

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (P450). We examined rat, rabbit, and human hepatic microsomal samples for their ability to activate ellipticine. The extent of activation was determined by binding of 3H-labeled ellipticine to DNA and by analyzing DNA adducts by 32P-postlabeling. We demonstrate that cytochrome P450 of human hepatic microsomes activating ellipticine to species binding to DNA is analogous to that of rats, but not of rabbits. Most of the ellipticine activation in rat and human hepatic microsomes is attributed to P450 enzymes of the same subfamily, P450 3A1/2 and P450 3A4, respectively, while the orthologous enzyme in rabbit hepatic microsomes, P450 3A6, is much less efficient. With purified enzymes, the major role of P450 3A1 and 3A4 in ellipticine-DNA adduct formation was confirmed. We identified deoxyguanosine as the target for P450-mediated ellipticine binding to DNA using polydeoxyribonucleotides and deoxyguanosine 3'-monophosphate. The results strongly suggest that rats are more suitable models than rabbits mimicking the metabolic activation of ellipticine in humans.
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PMID:Rat microsomes activating the anticancer drug ellipticine to species covalently binding to deoxyguanosine in DNA are a suitable model mimicking ellipticine bioactivation in humans. 1269 29

Ellipticine is a potent antineoplastic agent whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts in vitro and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). Here, we investigated the capacity of ellipticine to form DNA adducts in vivo. Male Wistar rats were treated with ellipticine, and DNA from various organs was analyzed by (32)P postlabeling. Ellipticine-specific DNA adduct patterns, similar to those found in vitro, were detected in most test organs. Only DNA of testes was free of the ellipticine-DNA adducts. The highest level of DNA adducts was found in liver (19.7 adducts per 10(7) nucleotides), followed by spleen, lung, kidney, heart and brain. One major and one minor ellipticine-DNA adducts were found in DNA of all these organs of rats exposed to ellipticine. Besides these, 2 or 3 additional adducts were detected in DNA of liver, kidney, lung and heart. The predominant adduct formed in rat tissues in vivo was identical to the deoxyguanosine adduct generated in DNA by ellipticine in vitro as shown by cochromatography in 2 independent systems. Correlation studies showed that the formation of this major DNA adduct in vivo is mediated by CYP3A1- and CYP1A-dependent reactions. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in vivo and confirm the formation of covalent DNA adducts as a new mode of ellipticine action.
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PMID:DNA adduct formation by the anticancer drug ellipticine in rats determined by 32P postlabeling. 1460 Oct 46

Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. To be useful for risk assessment, however, microarray data must be challenged to determine reliability and interlaboratory reproducibility. As part of a series of studies conducted by the International Life Sciences Institute Health and Environmental Science Institute Technical Committee on the Application of Genomics to Mechanism-Based Risk Assessment, the biological response in rats to the hepatotoxin clofibrate was investigated. Animals were treated with high (250 mg/kg/day) or low (25 mg/kg/day) doses for 1, 3, or 7 days in two laboratories. Clinical chemistry parameters were measured, livers removed for histopathological assessment, and gene expression analysis was conducted using cDNA arrays. Expression changes in genes involved in fatty acid metabolism (e.g., acyl-CoA oxidase), cell proliferation (e.g., topoisomerase II-Alpha), and fatty acid oxidation (e.g., cytochrome P450 4A1), consistent with the mechanism of clofibrate hepatotoxicity, were detected. Observed differences in gene expression levels correlated with the level of biological response induced in the two in vivo studies. Generally, there was a high level of concordance between the gene expression profiles generated from pooled and individual RNA samples. Quantitative real-time polymerase chain reaction was used to confirm modulations for a number of peroxisome proliferator marker genes. Though the results indicate some variability in the quantitative nature of the microarray data, this appears due largely to differences in experimental and data analysis procedures used within each laboratory. In summary, this study demonstrates the potential for gene expression profiling to identify toxic hazards by the identification of mechanistically relevant markers of toxicity.
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PMID:Clofibrate-induced gene expression changes in rat liver: a cross-laboratory analysis using membrane cDNA arrays. 1503 92


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