MOLECULAR MECHANISMS OF ANTINEOPLASTIC ACTION OF AN ANTICANCER DRUG ELLIPTICINE

Ellipticine is a potent antineoplastic agent exhibiting the multimodal mechanism of its action. This article reviews the mechanisms of predominant pharmacological and cytotoxic effects of ellipticine and shows the results of our laboratories indicating a novel mechanism of its action. The prevalent mechanisms of ellipticine antitumor, mutagenic and cytotoxic activities were suggested to be intercalation into DNA and inhibition of DNA topoisomerase II activity. We demonstrated a new mode of ellipticine action, formation of covalent DNA adducts mediated by its oxidation with cytochromes P450 (CYP) and peroxidases. The article reports the molecular mechanism of ellipticine oxidation by CYPs and identifies human and rat CYPs responsible for ellipticine metabolic activation and detoxication. It also presents a role of peroxidases (i.e. myeloperoxidase, cyclooxygenases, lactoperoxidase) in ellipticine oxidation leading to ellipticine-DNA adducts. The 9-hydroxyand 7-hydroxyellipticine metabolites formed by CYPs and the major product of ellipticine oxidation by peroxidases, the dimer, in which the two ellipticine skeletons are connected via N of the pyrrole ring of one ellipticine molecule and C9 in the second one, are the detoxication metabolites. On the contrary, 13-hydroxyand 12-hydroxyellipticine, produced by ellipticine oxidation with CYPs, the latter one formed also spontaneously from another CYPand peroxidase-mediated metabolite, ellipticine N-oxide, are metabolites responsible for formation of two ellipticine-derived deoxyguanosine adducts in DNA. The results reviewed here allow us to propose species, two carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, as reactive species generating two 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.


INTRODUCTION
Ellipticine (5,11-dimethyl-6H-pyrido [4,3-b]carbazole), an alkaloid isolated from Apocyanaceae plants (i.e. Ochrosia borbonica, Excavatia coccinea), and several its derivatives exhibit significant antitumor and anti-HIV activities [1][2][3] . This compound is one of the simplest naturally occurring alkaloids, having a planar structure 4 . It was first isolated in 1959 from the leaves of the evergreen tree Ochrosia elliptica, which grows wild in Oceania 4 . Ellipticine and 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 for the treatment of osteolytic breast cancer metastases, kidney cancer, brain tumors and acute myeloblastic leukemia [5][6][7][8][9] . The main reason for the interest in ellipticine and its derivatives for clinical purposes is their high efficiencies against several types of cancer, their rather limited toxic side effects and their complete lack of hematological toxicity 5 . Nevertheless, mutagenicity of ellipticines should be evaluated as a potential risk factor for these anticancer agents. Most ellipticines are mutagenic to Salmonella typhimurium Ames tester strains, bacteri-ophage T4, Neurospora crassa, and mammalian cells and induce prophage lambda in Escherichia coli 10 .

MECHANISMS OF THE CYTOTOXICITY AND ANTICANCER ACTIVITY OF ELLIPTICINES
Ellipticines are cytostatics, whose precise mechanisms of action have not yet been explained. The multimodal mechanism of their action was demonstrated 5 . Therefore, ellipticine antineoplastic activity may result from alternative cytotoxic events. It was suggested that the prevalent mechanisms of ellipticine antitumor, mutagenic and cytotoxic activities are (i) intercalation into DNA and (ii) inhibition of DNA topoisomerase II activity 5 .
The size and shape of the ellipticine chromophore closely resemble those of a purine-pyrimidine complementary base pair, providing favorable conditions for its intercalation in double-stranded DNA. Furthermore, the polycyclic aromatic character of the molecule may, moreover, result in tight interactions with appropriately conformed hydrophobic regions in DNA. Interactions between the methyl groups of the drug and the thymine bases at the intercalation site appear important in determining the orientational preferences of the drug [11][12] .
The mechanism of ellipticine action as the inhibitor of topoisomerase II has also been extensively studied [13][14][15] . Ellipticine acts by stimulating topoisomerase II-mediated DNA breakage. It is likely that formation of a ternary complex between topoisomerase II, DNA, and drug is critical for nucleic acid breakage and subsequent cell death. Topoisomerase II was identified as the primary cellular target of the drug. Furthermore, ellipticine did not inhibit enzyme-mediated DNA religation, suggesting that it stimulates DNA breakage by enhancing the forward rate of cleavage. Froelich-Ammon and coworkers 15 postulated that ellipticine enters the ternary complex through its prior association with either DNA or the enzyme and does not require the presence of a preformed topoisomerase II-DNA complex.
Numerous studies have reported the involvement of p53 tumor suppressor protein in ellipticine cytotoxic effect [16][17][18][19] . 9-Hydroxyellipticine treatment caused induction of apoptosis in the G1 phase of the cell cycle in mutant p53 transfected Saos-2 cells, but not in p53-deficient parental Saos-2 cells 17 . Ellipticine and 9-hydroxyellipticine cause selective inhibition of p53 protein phosphorylation via kinase inhibition in several human cancer cell lines such as Lewis lung carcinoma and human colon cancer cell line SW480 18 , and this correlated with their cytotoxic activity. Moreover, accumulation of dephosphorylated mutant p53 may induce apoptosis 18 . Ellipticine has also been found to restore the transcription function of mutant p53, and this property may contribute to resisting tumor cell lines expression mutant 16 . Ellipticine causes an increase in the protein expression of p53 and, p21/WAF1 and KIP1/p27, but not of WAF1/p21 in human breast adenocarcinoma MCF-7 cells treated with ellipticine, and growth inhibition by this compound is also induced by mitochondrial proapoptotic pathways in these cells 19 . In addition, ellipticines uncouple mitochondrial oxidative phosphorylation 20 , and thereby disrupt the energy balance of cells.
Studies on the mechanisms of the cytotoxic and anticancer activity of ellipticine have also shown that these activities might be due to induction of endoplasmic reticulum stress 21 . It is evident that the explanations of anticancer activity mentioned above are based on mechanisms of nonspecific drug actions. The transport of highly hydrophobic ellipticine molecules across cell membranes into cells (including both tumor and healthy cells) is also nonspecific. However, this sharply contrasts with specificity of antineoplastic activity of ellipticines against only several cancer diseases. The specificity of antitumor activity of ellipticines should, hence, be a consequence of other mechanisms of their action, which have not been elucidated as yet.

Oxidation of ellipticine by cytochromes P450 plays a role both in its detoxication and in activation to species binding to DNA
We have demonstrated that CYP enzymes expressed in human and rat livers are effective in activating ellipticine leading to species forming the covalent DNA adducts in vitro (see Fig. 1 for formation of ellipticine-derived DNA adducts by different concentrations of human CYP3A4).
The structure of all ellipticine metabolites produced by CYPs was characterized, the participation of individual human and rat CYP enzymes in their formation identified and the metabolites responsible for DNA binding elucidated 27,31 . HPLC was found to be suitable method for separation of individual ellipticine metabolites (Fig. 2). Four of ellipticine metabolites were identified to be C-hydroxylated derivatives of ellipticine, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-and 13-hydroxyellipticine and one is the N 2oxide of ellipticine (Figs. 2 and 3) (ref. 27,31 ). Employing three experimental approaches [correlation of CYP-linked enzyme activities in human hepatic microsomes with the amounts of ellipticine metabolites generated by the same microsomes, the effect of selective CYP inhibitors, and use of heterologous baculovirus expression systems of human CYPs (Supersomes TM )] CYP enzymes responsible for the oxidation of ellipticine in human hepatic microsomes were resolved 27,31 .
The formation of 9-hydroxy and 7-hydroxyellipticine is attributable to the activity of CYP1A1/2 as follows from correlation tendencies of their formation with ethoxyresorufin O-deethylase activity, a marker for CYP1A1/2 (Tab. 1). Furthermore, α-naphthophlavone and furafylline, selective CYP1A1/2 inhibitors, decreased formation of these metabolites efficiently 27,31 . The formation of 13-hydroxyellipticine was highly correlated with 6β-hydroxylation of testosterone, a marker for CYP3A4. A significant correlation was also seen between production of N 2 -oxide of ellipticine and 6β-hydroxylation of testosterone 27 . These results indicate that 13-hydroxyellipticine and N 2oxide of ellipticine are mainly generated by CYP3A4 in Data used for correlation analysis are shown in our previous work 27 . Statistical association between CYP-linked catalytic activities in human hepatic microsomal samples and levels of individual ellipticine metabolites or of ellipticine-DNA adducts formed by the same microsomes were determined by the correlation coefficients using version 6.12 Statistical Analysis System software. Correlation coefficients were based on a sample size of 8. All Ps are two-tailed and considered significant at the 0.05 level.
human livers (Tab. 1). Indeed ketoconazole, an inhibitor of CYP3A4 inhibited generation of 13-hydroxyellipticine and ellipticine N 2 -oxide significantly 31 . Utilizing microsomes of Baculovirus transfected insect cells (Supersomes TM ) containing recombinantly expressed human CYPs and NADPH:CYP reductase corroborated the results obtained with human hepatic microsomes, except for very effective N 2 -oxidation by CYP2D6 in this system 27 (Fig. 4). Human recombinant CYP1A1 and 1A2 were the major enzymes oxidizing ellipticine to 9-hydroxyand 7-hydroxyellipticine. Moreover, CYP1B1 and 2D6 were also efficient to catalyze these reactions, but to a lower extent. 12-Hydroxyellipticine was found to be a minor product of ellipticine oxidation by human recombinant CYP enzymes. Low levels of this metabolite are generated only in incubations containing CYP3A4 and 2C9. The major enzyme oxidizing ellipticine to 13-hydroxyellipticine is human recombinant CYP3A4, followed by CYP1A2, 2D6 and 2C9. The human recombinant CYP3A4 enzyme is also able to form the metabolite N 2 -oxide of ellipticine, but human recombinant CYP2D6 is much more effective in generating this product under the conditions used (Fig. 4). Because CYP3A4 is the most abundant CYP species in human liver (∼30 %) (ref. 32 ), oxidation of ellipticine to 13-hydroxyellipticine and N 2 -oxide of ellipticine should be the major metabolic pathway of the drug in human livers. Indeed, the predominant ellipticine metabolite formed by the human hepatic microsomal samples is 13-hydroxyellipticine followed by N 2 -oxide of ellipticine 27 . The CYP enzymes analogous to those of human livers were found to be responsible for oxidation of ellipticine in rat hepatic microsomes. 7-Hydroxy-and 9-hydroxyellipticine are generated mainly by rat CYP1A1/2, while 13-hydroxyellipticine and ellipticine N 2 -oxide by CYP3A1. Similarities in pattern of ellipticine metabolites and CYP enzymes responsible for their generation in human and rat hepatic microsomal systems underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine 24,27,31 . Therefore, rats were used as model organisms to study the fate of ellipticine in vivo 25 .
Using 32 P-postlabeling method 33 we found that during the ellipticine oxidation by CYPs two major and several minor adducts are generated in DNA (Fig. 1). Employing a panel of different human recombinant CYP enzymes, CYP3A4, 1A1 and 1B1, which are expressed at higher levels in tumors sensitive to ellipticine (i.e. breast and kidney cancer) than in peritumoral tissues [34][35][36][37] , were found to most efficiently activate ellipticine to form covalent DNA adducts in vitro 22 (Fig. 5). Deoxyguanosine was identified as the target base to which ellipticine metabolites generated by CYPs are bound 27 , forming the two major ellipticine-derived DNA adducts (adduct spots 1 and 2 in Fig. 1) in vitro 22,24,27 . These deoxyguanosine adducts with ellipticine were detected in human hepatic 27 and renal microsomes 30 , in V79 Chinese hamster lung fibroblasts transfected with human CYP3A4, 1A1 and 1A2 23 , in human breast adenocarcinoma MCF-7 cells 26 , in human HL-60 leukemia cells 28 and in several organs such as liver, kidney, lung, spleen, heart and brain of rats exposed to this anticancer drug 25 . Since CYP3A4 enzymatic activity     correlated with levels of these DNA adducts formed in human hepatic microsomes, CYP3A4 seems to be the major enzyme generating these DNA adducts in human livers ( Table 1). The orthologous rat enzymes, CYP3A1 and, furthermore, CYP1A1/2 are the enzymes responsible for DNA adducts formation in rats treated with ellipticine 25 . Besides these two major adducts, up to five minor adduct spots were detectable in DNA reacted with ellipticine activated with CYPs (ref. 22,27 ) or in V79 (ref. 23 ), MCF-7 (ref. 26 ) and HL-60 cells 28 exposed to ellipticine or in vivo 25 ( Fig. 6A f ). At the present time, it is not possible to demonstrate if the antitumor, cytostatic and/or genotoxic activities of ellipticine are related to only one or several of the DNA damage effects. Recently, we demonstrated that the cytotoxicity elicited by ellipticine towards Chinese hamster fibroblast V79 cells does not seem to be dependent on CYP expression 23 . Acute toxicity to these cells could be caused by the parent compound or by CYP independent metabolites. For ellipticine antitumour activity to cancer cells, however, other properties such as mutagenicity caused by DNA adducts, might be relevant. Indeed, Rekha and Sladek 38 demonstrated that antineoplastic activity of ellipticine to MCF-7 cells depends on the levels of CYP enzymes activating ellipticine to DNA-binding species. These authors showed that MCF-7 cells treated with 3methylcholanthrene transiently expressed elevated levels of CYP1A and cells were transiently much more sensitive to ellipticine. CYP1A also activate ellipticine to species binding to DNA [22][23][24] and in an earlier study we found the typical ellipticine-DNA adducts in these cells 26 . The CYPdependent higher sensitivity of MCF-7 cells to ellipticine observed by these authors might, therefore, be explained by ellipticine-DNA adduct formation.
Another important feature relating the expression of CYP and the antineoplastic activity of ellipticine was detected in MCF-7 cells selected for resistance to adriamycin (Adr R MCF-7) and exhibiting the phenotype of multidrug resistance (MDR) (ref. 39 ). Ivy et al. 39 postulated that the MDR property of Adr R MCF-7 cells involves several biochemical and genetic changes. One of them is a regulatory defect at the level of CYP1A1 mRNA resulting in lower CYP1A1-mediated metabolism of xenobiotics in these cells. Adr R MCF-7 cells are cross-resistant to ellipticine 39 , which we would explain by a decrease in the CYP1A1dependent activation of ellipticine. Taken together, the activities and expression levels of CYP enzymes, which effectively activate ellipticine to metabolites forming DNA adducts, may be important factors in the specificity of ellipticine for breast cancer. Nevertheless, to confirm this suggestion, formation of ellipticine-DNA adducts in breast cancer tissues in vivo remains to be evaluated. Preliminary results indicate that ellipticine-derived DNA adducts are detectable not only in healthy organs of rats exposed to ellipticine 25 , but also in the target tissue for the treatment, mammary tumors (Stiborová et al., unpublished results). Furthermore, to better understand the role of ellipticine-DNA adducts in the pharmacological efficacy in the cancer treatment or in the genotoxic side effects of the drug, we are analyzing the dose dependence and the persistence of ellipticine-DNA adducts in target and non-target tissues of rats treated with ellipticine. Because ellipticine is used in the treatment of breast cancer, differences in adduct patterns in the two sexes are also under investigation. 9-Hydroxyellipticine, the metabolite excreted in urine by humans (mainly in the form of conjugates), and 7hydroxyellipticine, were supposed to be the ellipticine detoxication products 5 . Indeed, their formation does not lead to generation of DNA adducts found in vivo, in rats treated with ellipticine [25][26][27] . On the contrary, we demonstrated that 13-hydroxy-, 12-hydroxyellipticine and the N 2 -oxide of ellipticine, are the metabolites generating the two major deoxyguanosine adducts in DNA in vitro and in vivo of several organs of rats treated with ellipticine. The identities of these deoxyguanosine adducts formed from 13-hydroxy-and 12-hydroxyellipticine with those formed in vitro using CYP-dependent activation systems and in rats in vivo was proved by co-chromatography on TLC and HPLC [23][24][25][26][27]30 (see also Fig. 6 showing the TLC and HPLC profiles of these two adducts). Therefore, 13-hydroxy-and 12-hydroxyellipticine are the products of the activation pathway of ellipticine metabolism. 12-Hydroxyellipticine is formed in two ways, one by direct oxidation, and one by Polonowski rearrangement 40,41 of ellipticine N 2 -oxide 27 . Therefore, the formation of 12-hydroxyellipticine or ellipticine N 2 -oxide, followed by its spontaneous rearrangement to 12-hydroxyellipticine, are two pathways leading to the formation of the same reactive species binding to DNA. Indeed, the production of ellipticine N 2 -oxide in human hepatic microsomes correlated with levels of DNA adduct 2 formation (Table 1).  25 ), and in some target cancers like breast and kidney cancer [34][35][36][37] , their levels are much lower in some other tissues or tumor cells (i.e. leukemia cells) that are sensitive to ellipticine. Indeed, ellipticine is cytotoxic to HL-60 promyelocytic leukemia cells and generates the DNA adducts in these cells 28 even though they express low levels of CYP 42 . The question thus arises, which other enzymes are involved in ellipticine activation in these malignant tissues. Peroxidases expressed in some cancer cells might be good candidates for such ellipticine activation 42-53 . In the mid 1980s the group of B. Meunier studied binding of one radioactively labeled ellipticine derivative, the antitumour drug 9-hydroxy-N 2 -methylellipticinium to DNA or RNA upon oxidation by horseradish peroxidase (HRP) 5, 54-56 . They and others proposed a mechanism by which this and other 9-hydroxylated ellipticine derivatives 5, 54-60 can act as bioalkylating agents [60][61][62][63] . These compounds are oxidized to the quinone-imine which then undergoes regiospecific Michael-type addition of bionucleophiles at the 10-position of quinone-imine yielding the adduct product with nucleophiles. Such adducts have been chemically prepared by oxidation of 9-hydroxyellipticine in the presence of nucleophiles by Meunier's 59,61 and Potier's 62, 63 research groups. The non-hydroxylated ellipticine derivatives were found by these authors not to be oxidized and to bind only very weakly to DNA 54 . In contrast to these results, our studies indicate that ellipticine is easily and effectively oxidized by peroxidases to species that bind to DNA [28][29][30] . Peroxidases such as bovine lactoperoxidase (LPO), human myeloperoxidase (MPO), ovine cyclooxygenase (COX)-1, human COX-2 and plant HRP oxidize ellipticine to species binding to DNA. Such activation was also found in incubations with one human renal microsomal sample in the presence of arachidonic acid, a cofactor of COX 30 . Using two independent methods, 32 P-postlabeling and 3 H-labeled ellipticine, we showed that ellipticine binds covalently to DNA in vitro after its oxidation by the peroxidases. During the elllipticine oxidation by peroxidases in vitro, two ellipticine metabolites were detectable under the conditions used in our experiments (Fig. 6). The major one was characterized by NMR spectroscopy as the ellipticine dimer, where two ellipticine residues are connected via the nitrogen atom N 6 in the pyrrole ring of one of the ellipticine molecules and the carbon atom C9 of the second ellipticine 30 (Fig. 7). The mechanism by which the ellipticine dimer is generated was resolved 29,30 . We showed that peroxidases metabolize ellipticine in a one-electron oxidation to free radicals, which, depending on the environment, generate either the additional metabolites such as ellipticine dimer or DNA adducts. Taking the structure of the dimer into account (Fig. 7), the first radical would be on the secondary nitrogen atom of the pyrrole ring (N 6 ). It should be noted that the dimer product is much less likely to be formed physiologically than in this experimental system, because no other molecules (bionucleophiles) are present there to intercept ellipticine reactive intermediates. Indeed, during the ellipticine oxidation by peroxidases in the presence of DNA up to four DNA adducts are formed, and an increase in the formation of the major DNA adduct 1 correlates with a decrease in generation of the ellipticine dimer 30 . Hence, ellipticine-DNA adduct formation seems to be the preferential reaction under the physiological conditions. Even though a two-electron-oxidation metabolite such as methylene-imine ( Fig. 7) was not found to be formed from ellipticine under the conditions used in our experiments 29,30 , we still suggest that such an ellipticine metabolite is responsible for the formation of the major ellipticine-DNA adduct (spot 1 in Figs. 1 and 6). This suggestion is strongly supported by our finding that the optimal adduct 1 formation required stoichiometric equivalents of ellipticine and oxidizing co-substrate, hydrogen peroxidase, during the peroxidase-mediated oxidation of ellipticine 30 .
As shown in Fig. 6, this adduct is generated from 13hydroxyellipticine 27 The exact reactive species as well as the positions in guanine where the reactive species generated from 13-hydroxyellipticine are bound remain to be elucidated. Nevertheless, we have suggested 27 that 13hydroxyellipticine might, depending on the environment, decompose spontaneously to the reactive carbenium ion (Fig. 7), which reacts with one of the nucleophilic centers in the deoxyguanosine residue in DNA (i.e. the exocyclic -NH 2 group of guanine). The identity of the adduct 1 generated from 13-hydroxyellipticine with that formed by peroxidases allow us to estimate the structure of a two-electron oxidation metabolite of ellipticine responsible to its formation as the methylene-imine derivative of ellipticine (Fig. 7). Such a reactive compound would generate an identical carbenium ion as 13-hydroxyellipticine (ellipticine-13-ylium, Fig. 7). In other words, 13-hydroxyellipticine acts as precursor of the vinylogous imine intermediate (methylene-imine) (Fig. 7), Michael-type addition of nucleophiles to this vinylogous imine then yields an adduct identical to that formed by the carbenium ion 64 .
The minor ellipticine oxidation product formed by peroxidases is the ellipticine N 2 -oxide, thus, the same metabolite that is also generated by human CYP enzymes 27 . As mentioned above, an N 2 -oxidation of ellipticine was found to be the prerequisite for the formation of 12-hydroxyellipticine metabolite (by the Polonowski rearrangement 27,43,44 ), which is responsible for generation of the second deoxyguanosine adduct in DNA (spot 2 in Figs. 1 and 6) 27 . Indeed, the identical DNA adduct was generated from ellipticine after its activation by peroxidases. As in the case of 13-hydroxyellipticine, we postulate another carbenium ion reacting with the nucleophillic centers of deoxyguanosine in DNA, generating the DNA adduct 2 (Fig. 7).
Two additional adducts (spots 6 and 7 in Fig. 6) formed in DNA by ellipticine activated with peroxidases were also observed in incubations with human hepatic 27 and renal microsomes 30 and in DNA of several tissues of rats treated with ellipticine 25 Peroxidase oxidation of ellipticine leads to much higher levels of these adducts than are formed in vivo 25 or in incubations with hepatic microsomes 27 Other minor ellipticine-DNA adducts found in vivo (spots 3-5 in Fig. 6), however, were not detectable in the peroxidase activation system. The mechanism of formation of these minor adducts and their structural characterization remains to be investigated.
Although HRP might only serve as a model peroxidase, the findings that MPO, LPO, COX-1 and COX-2 are effective in ellipticine oxidation may be of greater significance. Human MPO is expressed in acute myeloblastic leukemia 43,53 , and might be involved in metabolic activation of drugs including ellipticine in leukemic myeloblasts 42,52 . Because of the high expression of this enzyme in acute myeloblastic leukemia, it is also used as a diagnostic marker in this leukemia. In addition, human MPO expressed in neutrophils and present in milk and blood 46, 56 might, besides CYPs, participate in metabolism of ellipticine in other cancers 42,52 . Likewise, LPO is secreted by human mammary ductal epithelial cells into the breast duct 46 and might metabolize anticancer drugs including ellipticine in other cancers 65 . Namely, like other lipophilic compounds ellipticine may accumulate in fatty tissues, such as the breast, and depending on the levels of activating enzymes present (e.g. LPO and MPO) ellipticine can be oxidized to intermediates modifying key molecules such as DNA in this tissue. Hydrogen peroxide, required for peroxidase-mediated oxidations, comes from respiratory burst of neutrophils and is also supplied by xanthine oxidase 65,66 .
The finding that ovine COX-1 and human COX-2 are capable of oxidative activation of ellipticine seems to have even greater significance. COX-1 is constitutively expressed with near constant levels and activity in many tissues and in several tumors (i.e. in brain tumors) 67 . Moreover, overexpression of COX-2 was demonstrated in multiple cancer types (i.e. carcinomas and brain tumors) and some pre-neoplastic lesions and is even inducible by carcinogenic processes and/or by several compounds, including anticancer drugs 44,[46][47][48][49][50] . Even though the effect of ellipticine on COX-2 expression has not yet been investigated, this peroxidase may play an important role in ellipticine metabolic activation in cancer cells, in which low levels of CYP enzymes are expressed. Therefore, a detailed study analyzing the relationships between lev-els of DNA-adducts formed from ellipticine activated by peroxidases, and expression levels of these enzymes and ellipticine cytotoxicity in human leukemia cells is under way in our laboratory. The objective to investigate the participation of peroxidases in the metabolic activation of ellipticine in vivo is our next goal. For instance, MPOknockout mice may help to evaluate the involvement of this enzyme in the bioactivation of ellipticine in vivo 68 .