Neuroblastoma (NBL) originates from undifferentiated cells of the sympathetic nervous system. Chemotherapy is judged to be suitable for successful treatment of this disease. Here, the influence of histone deacetylase (HDAC) inhibitor valproate (VPA) combined with DNA-damaging chemotherapeutic, ellipticine, on UKF-NB-4 and SH-SY5Y neuroblastoma cells was investigated. Treatment of these cells with ellipticine in combination with VPA led to the synergism of their anticancer efficacy. The effect is more pronounced in the UKF-NB-4 cell line, the line with N-myc amplification, than in SH-SY5Y cells. This was associated with caspase-3-dependent induction of apoptosis in UKF-NB-4 cells. The increase in cytotoxicity of ellipticine in UKF-NB-4 by VPA is dictated by the sequence of drug administration; the increased cytotoxicity was seen only after either simultaneous exposure to these drugs or after pretreatment of cells with ellipticine before their treatment with VPA. The synergism of treatment of cells with VPA and ellipticine seems to be connected with increased acetylation of histones H3 and H4. Further, co-treatment of cells with ellipticine and VPA increased the formation of ellipticine-derived DNA adducts, which indicates an easier accessibility of ellipticine to DNA in cells by its co-treatment with VPA and also resulted in higher ellipticine cytotoxicity. The results are promising for in vivo studies and perhaps later for clinical studies of combined treatment of children suffering from high-risk NBL.

• Klíčová slova
• DNA damage, acetylation of histones, apoptosis, ellipticine, neuroblastoma, valproate,
• MeSH
• apoptóza MeSH
• elipticiny toxicita   MeSH
• inhibitory histondeacetylas toxicita   MeSH
• kyselina valproová toxicita   MeSH
• lidé MeSH
• mutageny toxicita   MeSH
• nádorové buněčné linie MeSH
• neuroblastom metabolismus   MeSH
• neurony účinky léků   metabolismus   MeSH
• synergismus léků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• inhibitory histondeacetylas MeSH
• kyselina valproová MeSH
• mutageny MeSH

ABSTRACT: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase.

• Klíčová slova
• Coenzymes, DNA, Enzymes, High pressure liquid chromatography,
• Publikační typ
• časopisecké články MeSH

ABSTRACT: Cytochrome P450 (CYP) 2S1 is "orphan" CYP that is overexpressed in several epithelial tissues and many human tumors. The pure enzyme is required for better understanding of its biological functions. Therefore, human CYP2S1 was considered to be prepared by the gene manipulations and heterologous expression in Escherichia coli. Here, the conditions suitable for efficient expression of human CYP2S1 protein from plasmid pCW containing the human CYP2S1 gene were optimized and the enzyme purified to homogeneity. The identity of CYP2S1 as the product of heterologous expression was confirmed by dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. To confirm the presence of the enzymatically active CYP2S1, the CO spectrum of purified CYP2S1 was recorded. Since CYP2S1 was shown to catalyze oxidation of compounds having polycyclic aromatic structures, the prepared enzyme has been tested to metabolize the compounds having this structural character; namely, the human carcinogen benzo[a]pyrene (BaP), its 7,8-dihydrodiol derivative, and an anticancer drug ellipticine. Reaction mixtures contained besides the test compounds the CYP2S1 enzyme reconstituted with NADPH:CYP reductase (POR) in liposomes, and/or this CYP in the presence of cumene hydroperoxide or hydrogen peroxide. High performance liquid chromatography was employed for separation of BaP, BaP-7,8-dihydrodiol, and ellipticine metabolites. The results found in this study demonstrate that CYP2S1 in the presence of cumene hydroperoxide or hydrogen peroxide catalyzes oxidation of two of the test xenobiotics, a metabolite of BaP, BaP-7,8-dihydrodiol, and ellipticine. Whereas BaP-7,8,9,10-tetrahydrotetrol was formed as a product of BaP-7,8-dihydrodiol oxidation, ellipticine was oxidized to 12-hydroxyellipticine, 13-hydroxyellipticine, and the ellipticine N2-oxide.

• Klíčová slova
• Coenzymes, Enzymes, High pressure liquid chromatography,
• Publikační typ
• časopisecké články MeSH

Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

• MeSH
• cytochrom P-450 CYP1A1 nedostatek   genetika   metabolismus   MeSH
• elipticiny farmakologie   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• myši MeSH
• poškození DNA * MeSH
• protinádorové látky farmakologie   MeSH
• rozpřahující látky farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• cytochrom P-450 CYP1A1 MeSH
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• protinádorové látky MeSH
• rozpřahující látky MeSH

The requirements for early diagnostics as well as effective treatment of cancer diseases have increased the pressure on development of efficient methods for targeted drug delivery as well as imaging of the treatment success. One of the most recent approaches covering the drug delivery aspects is benefitting from the unique properties of nanomaterials. Ellipticine and its derivatives are efficient anticancer compounds that function through multiple mechanisms. Formation of covalent DNA adducts after ellipticine enzymatic activation is one of the most important mechanisms of its pharmacological action. In this study, we investigated whether ellipticine might be released from its micellar (encapsulated) form to generate covalent adducts analogous to those formed by free ellipticine. The (32)P-postlabeling technique was used as a useful imaging method to detect and quantify covalent ellipticine-derived DNA adducts. We compared the efficiencies of free ellipticine and its micellar form (the poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO) block copolymer, P 119 nanoparticles) to form ellipticine-DNA adducts in rats in vivo. Here, we demonstrate for the first time that treatment of rats with ellipticine in micelles resulted in formation of ellipticine-derived DNA adducts in vivo and suggest that a gradual release of ellipticine from its micellar form might produce the enhanced permeation and retention effect of this ellipticine-micellar delivery system.

• MeSH
• adukty DNA chemie   metabolismus   MeSH
• elipticiny aplikace a dávkování   chemie   farmakokinetika   MeSH
• krysa rodu Rattus MeSH
• metabolická clearance MeSH
• micely MeSH
• orgánová specificita MeSH
• potkani Wistar MeSH
• příprava léků metody   MeSH
• protinádorové látky aplikace a dávkování   chemie   farmakokinetika   MeSH
• tkáňová distribuce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• micely MeSH
• protinádorové látky MeSH

Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. Here, a comparison of the toxicity of ellipticine to human breast adenocarcinoma MCF-7 cells, leukemia HL-60 and CCRF-CEM cells, neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cells and U87MG glioblastoma cells and mechanisms of its action to these cells were evaluated. Treatment of all cells tested with ellipticine resulted in inhibition of cell growth and proliferation. This effect was associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP and peroxidase enzymes, in MCF-7, HL-60, CCRF-CEM, UKF-NB-3, UKF-NB-4 and U87MG cells, but not in neuroblastoma UKF-NB-3 cells. Therefore, DNA adduct formation in most cancer cell lines tested in this comparative study might be the predominant cause of their sensitivity to ellipticine treatment, whereas other mechanisms of ellipticine action also contribute to its cytotoxicity to neuroblastoma UKF-NB-3 cells.

• Klíčová slova
• DNA adducts, Ellipticine, cancer cell lines, mechanims of acticancer effects of ellipticine,
• Publikační typ
• časopisecké články MeSH

Two compounds known to covalently bind to DNA after their activation with cytochromes P450 (CYPs), carcinogenic benzo(a)pyrene (BaP) and an antineoplastic agent ellipticine, were investigated for their potential to induce CYP and NADPH:CYP reductase (POR) enzymes in rodent livers, the main target organ for DNA adduct formation. Two animal models were used in the study: (i) rats as animals mimicking the fate of ellipticine in humans and (ii) mice, especially wild-type (WT) and hepatic POR null (HRN™) mouse lines. Ellipticine and BaP induce expression of CYP1A enzymes in livers of experimental models, which leads to increase in their enzymatic activity. In addition, both compounds are capable of generating DNA adducts, predominantly in livers of studied organisms. As determined by (32)P postlabelling analysis, levels of ellipticine-derived DNA adducts formed in vivo in the livers of HRN™ mice were reduced (by up to 65%) relative to levels in WT mice, indicating that POR mediated CYP enzyme activity is important for the activation of ellipticine. In contrast to these results, 6.4 fold higher DNA binding of BaP was observed in the livers of HRN™ mice than in WT mice. This finding suggests a detoxication role of CYP1A in BaP metabolism in vivo. In in vitro experiments, DNA adduct formation in calf thymus DNA was up to 25 fold higher in incubations of ellipticine or BaP with microsomes from pretreated animals than with controls. This stimulation effect was attributed to induction of CYP1A1/2 enzymes, which are responsible for oxidative activation of both compounds to the metabolites generating major DNA adducts in vitro. Taken together, these results demonstrate that by inducing CYP1A1/2, ellipticine and BaP modulate their own enzymatic metabolic activation and detoxication, thereby modulating their either pharmacological (ellipticine) and/or genotoxic potential (both compounds).

• Klíčová slova
• HRN™ mice, NADPH:cytochrome P450 reductase, benzo(a)pyrene, cytochromes P450, ellipticine, induction,
• Publikační typ
• časopisecké články MeSH