Mammalian peroxidases activate anticancer drug ellipticine to intermediates forming deoxyguanosine adducts in DNA identical to those found in vivo and generated from 12‐hydroxyellipticine and 13‐hydroxyellipticine

Abstract

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation, inhibition of topoisomerase II and cytochrome P450-mediated formation of covalent DNA adducts. This is the first report on the molecular mechanism of ellipticine oxidation by peroxidases (human myeloperoxidase, human and ovine cyclooxygenases, bovine lactoperoxidase, horseradish peroxidase) to species forming ellipticine-DNA adducts. Using NMR spectroscopy, the structures of 2 ellipticine metabolites were identified; the major product is the ellipticine dimer, in which the 2 ellipticine skeletons are connected via N 6 of the pyrrole ring of one ellipticine molecule and C9 in the second one. The minor metabolite is ellipticine N 2 -oxide. Using 32 P-postlabeling and [ 3 H]-labeled ellipticine, we showed that ellipticine binds covalently to DNA after its activation by peroxidases. The DNA adduct pattern induced by ellipticine consisted of a cluster of up to 4 adducts. The 2 adducts are indistinguishable from the 2 major adducts generated between deoxyguanosine in DNA and either 13-hydroxy-or 12-hydroxyellipticine or in rats treated with ellipticine, or if ellipticine was activated with human hepatic and renal microsomes. The results presented here are the first characterization of the peroxidase-mediated oxidative metabolites of ellipticine and we have proposed species, 2 carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, as reactive species generating 2 major DNA adducts seen in vivo in rats treated with ellipticine. The study forms the basis to further predict the susceptibility of human cancers to ellipticine. ' 2006 Wiley-Liss, Inc.Key words: ellipticine; anticancer drug; peroxidase; cytochrome P450; DNA adduct Ellipticine (5,carbazole), an alkaloid isolated from Apocyanaceae plants and several of its more soluble derivatives (9-hydroxyellipticine, 9-hydroxy-N 2 -methylellipticinium, 9-chloro-N 2 -methylellipticinium and 9-methoxy-N 2 -methylellipticinium) exhibit promising results in the treatment of osteolytic breast cancer metastases, kidney cancer, brain tumors and acute myeloblastic leukemia (for a summary see literature 1 ). The main reason for the interest in ellipticine and its derivatives for clinical purposes is their high efficiency against several types of cancer, their rather limited toxic side effects and their complete lack of hematological toxicity. 2 Nevertheless, ellipticine is a potent mutagen. Most ellipticines are mutagenic to Salmonella typhimurium Ames tester strains, bacteriophage T4, Neurospora crassa and mammalian cells and induce prophage lambda in Escherichia coli (for an overview see literature 1 ).The antineoplastic property of ellipticine was considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. 3-7 Recently, we found another mode of ellipticine action. 1,[8][9][10] We demonstrated that ellipticine covalently binds to DNA after being enzymatically activated. Using a panel of different human recombinant cytochrome...

Analysis of covalent ellipticine- and doxorubicin-derived adducts in DNA of neuroblastoma cells by the ³²P-postlabeling technique

“…(Unterach, Austria), dimethyl sulfoxide (DMSO) from Amresco Inc. (Solon, OH, USA), phenolchloroform from Roth (Karlsruhe, Germany) and isopropanol from PLIVA-Lachema (Brno, Czech Republic). 12-Hydroxy-and 13-hydroxyellipticine were isolated from multiple high-performance liquid chromatography (HPLC) runs of ethyl acetate extracts of incubations containing ellipticine and human and/or rat hepatic microsomes as described 12,33,34 . All these and other chemicals used in the experiments were of analytical purity or better.…”

“…7 ), from calf thymus DNA reacted with 13-hydroxyellipticine (B) and 12-hydroxyellipticine (C) (ref. 33,34 ). Analyses were performed by the nuclease P1 version of the ellipticine for both neuroblastoma cell lines did not differ significantly, the UKF-NB-4 cell line (the less-aggressive S-type line) was less sensitive to this drug than the UKF-NB-3 cell line.…”

“…[29][30][31][32] ). However, whereas it is useful for detecting and quantifying covalent ellipticinederived DNA adducts 12,14,15,33,34 , its ability to determine covalent doxorubicin-DNA adducts remains to be resolved. The present study was therefore undertaken to investigate whether covalent doxorubicin-DNA adducts are detectable by the 32 P-postlabeling technique.…”

“…P-postlabeling of ellipticine-derived DNA adducts The 32 P-postlabeling of nucleotides using nuclease P1 enrichment procedure 31 , found previously to be appropriate to detect and quantify ellipticine-derived DNA adducts formed in vitro and in vivo 12,14,33,34 was used previously to detect and quantify the adducts formed in neuroblastoma cells 7 . DNA isolated from neuroblastoma cells as described in that study 7 and DNA from experiments performed earlier, namely, calf thymus DNA incubated with 13-hydroxy-and 12-hydroxyellipticine 33,34 were labeled with 32 P to show and compare adduct spot patterns.…”

“…DNA isolated from neuroblastoma cells as described in that study 7 and DNA from experiments performed earlier, namely, calf thymus DNA incubated with 13-hydroxy-and 12-hydroxyellipticine 33,34 were labeled with 32 P to show and compare adduct spot patterns.…”