Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N 2 -oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N 2 -oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N 2 -oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N 2 -oxide.
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...
Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the molecular mechanism of DNA-mediated ellipticine action in human neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cancer cell lines was investigated. Treatment of neuroblastoma cells with ellipticine resulted in apoptosis induction, which was verified by the appearance of DNA fragmentation, and in inhibition of cell growth. These effects were associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by the cytochrome P450-and peroxidase-mediated ellipticine metabolites, 13-hydroxy-and 12-hydroxyellipticine. The expression of these enzymes at mRNA and protein levels and their ability to generate ellipticine-DNA adducts in neuroblastoma cells were proven, using the real-time polymerase chain reaction, Western blotting analyses and by analyzing ellipticine-DNA adducts in incubations of this drug with neuroblastoma S9 fractions, enzyme cofactors and DNA. The levels of DNA adducts correlated with toxicity of ellipticine to IMR-32 and UKF-NB-4 cells, but not with that to UKF-NB-3 cells. In addition, hypoxic cell culture conditions resulted in a decrease in ellipticine toxicity to IMR-32 and UKF-NB-4 cells and this correlated with lower levels of DNA adducts. Both these cell lines accumulated in S phase, suggesting that ellipticine-DNA adducts interfere with DNA replication. The results demonstrate that among the multiple modes of ellipticine antitumor action, formation of covalent DNA adducts by ellipticine is the predominant mechanism of cytotoxicity to IMR-32 and UKF-NB-4 neuroblastoma cells.
Histone deacetylase (HDAC) inhibitors are a group of anticancer drugs which cause growth arrest and apoptosis of several tumor cells. HDAC inhibitors have been also found to increase the anticancer efficacy of several treatment modalities i.e. chemotherapy or radiotherapy. Here, we review the literature on combinations of HDAC inhibitors both with ionizing radiation and with other drugs, highlighting DNA-damaging compounds. The results of numerous studies with several types of cancer cells discussed in this review demonstrate that HDAC inhibitors enhance the effect of DNA damaging agents, such as inhibitors of topoisomerases, inhibitors of DNA synthesis, DNA-intercalators and agents covalently modifying DNA (i.e. doxorubicin, etoposid, 5-fluorouracil, cisplatin, melphalan, temozolomide and ellipticine) or of irradiation. Hence, the use of HDAC inhibitors combined with these antitumor drugs or ionizing radiation is a promising tool which may make treatment of patients suffering from many types of cancer more efficient. Several molecular mechanisms are responsible for the observed higher sensitivity of tumor cells towards therapeutic agents elicited by HDAC inhibitors. These mechanisms are discussed also in this review.
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