logo ban ban logo

Tajemství vládnutí spočívá totiž v tom, jak spojit víru ve vlastní neomylnost se schopností učit se z minulých chyb.

George Orwell

mendel

Výzkum

Expression Levels of Enzymes Metabolizing an Anticancer Drug Ellipticine Determined by Electromigration Assays Influence its Cytotoxicity to Cancer Cells - A Comparative Study

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole, Fig. 1) and its derivatives are efficient anticancer compounds that function through multiple mechanisms participating in cell cycle arrest and initiation of apoptosis (for a summary see [1-6]). Ellipticine was found (i) to arrest cell cycle progression due to modulation of levels of cyclinB1 and Cdc2, and phosphorylation of Cdc2 in human mammary adenocarcinoma MCF-7 cells, (ii) to initiate apoptosis due to formation of toxic free radicals, stimulation of the Fas/Fas ligand system and modulation of proteins of Bcl-2 family in several tumor cell lines, and (iii) to induce the mitochondria-dependent apoptotic processes (for a summary see [3,4]). The predominant mechanisms of ellipticine’s biological effects were suggested to be (i) intercalation into DNA [5-7] and (ii) inhibition of topoisomerase II [3-6]. Further, we showed that this antitumor agent forms covalent DNA adducts after its enzymatic activation with cytochromes P450 (CYP) and peroxidases [1-4,8-13], suggesting an additional DNA-damaging effect of ellipticine. Of the CYP enzymes investigated, human CYP3A4 followed by CYP1A1 and 1B1 are the most active enzymes oxidizing ellipticine to 12-hydroxy- and 13-hydroxyellipticine, the reactive metabolites that dissociate to ellipticine-12-ylium and ellipticine-13-ylium, which bind to DNA [3,7,9-11]. The CYP1A isoforms also efficiently form the other ellipticine metabolites, 7-hydroxy- and 9-hydroxyellipticine, which are the detoxification products (Fig. 1). Recently, we found that cytochrome b5 alters the ratio of ellipticine metabolites formed by CYP1A1, 1A2 and 3A4. While the amounts of the detoxification metabolites (7-hydroxy- and 9-hydroxyellipticine) were either decreased or not changed with added cytochrome b5, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N2-oxide increased considerably. The change in amounts of metabolites resulted in an increased formation of covalent ellipticine-DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action [11,12]. In addition, we observed that levels of the DNA adduct formed by 13-hydroxyellipticine also increased if this ellipticine metabolite was conjugated with sulfate or acetate by human sulfotransferases 1A1, 1A2, 1A3 and 2A1, or N,O-acetyltransferases 1 and 2 [11-14] as it is shown in Fig. 1. The same ellipticine-derived DNA adducts that were found in in-vitro incubations of ellipticine with DNA and enzymes activating this drug, were generated also in vivo, in several tissues of mice and rats exposed to ellipticine. In both animal models, ellipticine-DNA adduct formation was mediated mainly by CYP1A and 3A enzymes, but a role of peroxidases in several organs has been proved [15-17]. Therefore, expression levels of CYP and peroxidase enzymes metabolizing ellipticine seem to be crucial for antitumor, cytostatic and genotoxic activities of this drug in individual tissues. The ellipticine-DNA adducts were also found in several cancer cell lines and in DNA of rat mammary adenocarcinoma in vivo [3,19-25]. In this study, the utilizing the findings from several former studies [19-25] as well as new results found in this work, we evaluated whether cytotoxicity of ellipticine to cancer cells is dependent on formation of covalent ellipticine-DNA adducts and whether the expression of the enzymes metabolizing ellipticine in vitro and in vivo regulates their levels in cancer cells as well as the ellipticine cytotoxicity to these cells. The method of Western blotting determined the enzyme protein expression levels, whereas the 32P-postlabeling method detected and quantified DNA adducts formed by ellipticine [1-3,25-29].

Práce je spojená s projektem CYTORES GACR (grant P301/10/0356).

Zemědělská 1/1665
613 00 Brno
Budova D		Tel.: +420 545 133 350
Fax.: +420 545 212 044