Vacuolar-ATPase-mediated intracellular sequestration of ellipticine contributes to drug resistance in neuroblastoma cells.

Neuroblastoma is the most common cancer in infants and the fourth most common cancer in children. Aggressive cell growth and chemoresistance are notorious obstacles in neuroblastoma therapy. Exposure to the anticancer drug ellipticine inhibits efficiently growth of neuroblastoma cells and induces apoptosis in these cells. However, ellipticine induced resistance in these cells. The upregulation of a vacuolar (V)-ATPase gene is one of the factors associated with resistance development. In accordance with this finding, we found that levels of V-ATPase protein expression are higher in the ellipticine-resistant UKF-NB-4ELLI line than in the parental ellipticine-sensitive UKF-NB-4 cell line. Treatment of ellipticine-sensitive UKF-NB-4 and ellipticine-resistant UKF-NB-4ELLI cells with ellipticine-induced cytoplasmic vacuolization and ellipticine is concentrated in these vacuoles. Confocal microscopy and staining of the cells with a lysosomal marker suggested these vacuoles as lysosomes. Transmission electron microscopy and no effect of an autophagy inhibitor wortmannin ruled out autophagy. Pretreatment with a V-ATPase inhibitor bafilomycin A and/or the lysosomotropic drug chloroquine prior to ellipticine enhanced the ellipticine­mediated apoptosis and decreased ellipticine-resistance in UKF-NB-4ELLI cells. Moreover, pretreatment with these inhibitors increased formation of ellipticine-derived DNA adducts, one of the most important DNA-damaging mechanisms responsible for ellipticine cytotoxicity. In conclusion, resistance to ellipticine in the tested neuroblastoma cells is associated with V-ATPase-mediated vacuolar trapping of this drug, which may be decreased by bafilomycin A and/or chloroquine.

Hypoxia-mediated histone acetylation and expression of N-myc transcription factor dictate aggressiveness of neuroblastoma cells.

Cells of solid malignancies generally adapt to entire lack of oxygen. Hypoxia induces the expression of several genes, which allows the cells to survive. For DNA transcription, it is necessary that DNA structure is loosened. In addition to structural characteristics of DNA, its epigenetic alterations influence a proper DNA transcription. Since histones play a key role in epigenetics, changes in expression levels of acetylated histones H3 and H4 as well as of hypoxia-inducible factor-1α (HIF-1α) in human neuroblastoma cell lines cultivated under standard or hypoxic conditions (1% O2) were investigated. Moreover, the effect of hypoxia on the expression of two transcription factors, c-Myc and N-myc, was studied. Hypoxic stress increased levels of acetylated histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cells with N-myc amplification, whereas almost no changes in acetylation of these histones were found in an SK-N-AS neuroblastoma cell line, the line with diploid N-myc status. An increase in histone H4 acetylation caused by hypoxia in UKF-NB-3 and UKF-NB-4 corresponds to increased levels of N-myc transcription factor in these cells.

Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5.

Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine-DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b(5) on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine-DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6>2C19>1B1>1A2>2E1 and >2C9. Cytochrome b(5) increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9>3A4>2D6>2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N(2)-oxide. CYP3A4 and 1A1 in the presence of cytochrome b(5) are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine-DNA adduct 2. These ellipticine-DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC(50) value of ellipticine in these cells was 0.7 µM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

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

BACKGROUND: Ellipticine and doxorubicin are antineoplastic agents, whose action is based mainly on DNA damage such as intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts. The key target to resolve which of these mechanisms are responsible for ellipticine and doxorubicin anticancer effects is the development of suitable methods for identifying their individual DNA-damaging effects. Here, the (32)P-postlabeling method was tested to detect covalent DNA adducts formed by ellipticine and doxorubicin. METHODS: The standard procedure of (32)P-postlabeling assay, this procedure under ATP-deficient conditions, the version using extraction of adducts with n-butanol and the nuclease P1 enrichment version were used to analyze ellipticineand/ or doxorubicin-derived DNA adducts. RESULTS: Two covalent ellipticine-derived DNA adducts, which are associated with cytotoxicity of ellipticine to human UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines, were detected by the (32)P-postlabeling method. These adducts are identical to those formed by the ellipticine metabolites, 13-hydroxy- and 12-hydroxyellipticine. In contrast, no covalent adducts formed by doxorubicin in DNA of these neuroblastoma cells and in DNA incubated with this drug and formaldehyde in vitro were detectable by the (32)P-postlabeling assay. CONCLUSIONS: The results presented in this paper are the first to demonstrate that in contrast to covalent DNA adducts formed by ellipticine, the adducts generated by formaldehyde-mediated covalent binding of doxorubicin to DNA are not detectable by the (32)P-postlabeling assay. No DNA adducts were, detectable either in vitro, in incubations of DNA with doxorubicin or in DNA of neuroblastoma cells treated with this drug. The results also suggest that covalent binding of ellipticine to DNA of UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines is the predominant mechanism responsible for the cytotoxicity of this drug. To understand the mechanisms of doxorubicin anticancer effects on neuroblastoma cells, development of novel methods for identifying covalent doxorubicin-derived DNA adducts is the major challenge for further research.

Impact of histone deacetylase inhibitor valproic acid on the anticancer effect of etoposide on neuroblastoma cells.

OBJECTIVES: Etoposide (Vepesid, VP-16), an inhibitor of topoisomerase II, is a chemotherapeutic drug commonly used for treatment of different types of malignant diseases. By inhibiting the topoisomerase II enzyme activity in cancer cells, this drug leads to DNA damage and subsequently to cell death. In this study, we investigated the effect of this anticancer drug alone and in combination with a histone deacetylase (HDAC) inhibitor, valproic acid (VPA), on a human UKF-NB-4 neuroblastoma cell line. METHODS: The effects of etoposide and VPA on UKF-NB-4 cells were tested under the normoxic and also the hypoxic (1% O2) cultivation conditions. The cytotoxicity of etoposide and VPA to a UKF-NB-4 neuroblastoma cell line was evaluated with MTT assay. Apoptosis of the cells was analyzed by flow cytometry using an Annexin V and propidium iodide binding method. The effect of etoposide and VPA on the cell cycle distribution was determined by flow cytometric analysis using propidium iodide staining. RESULTS: The results of the study demonstrate that UKF-NB-4 neuroblastoma cells are sensitive both to etoposide and to VPA. They also indicate that the impact of VPA on cytotoxicity of etoposide in these tumor cells varies depending on the sequence of cultivation of the cells with the drugs. As a suitable sequence of cultivation, with a high rate of suppression of neuroblastoma cell growth was found the preincubation of the cells with etoposide, which was followed by their cultivation with VPA. In contrast, the reversed combination (preincubation of the cells with VPA before their treating with etoposide) did not give any increase in etoposide cytotoxicity. The effect of such combined treatment can be explained by measuring the cell cycle distribution, which shows that both etoposide and VPA change the cell cycle phase distribution. CONCLUSION: Etoposide and VPA were found as cycle phase specific drugs that are cytotoxic to human UKF-NB-4 neuroblastoma cells used either as single drugs or both together. However, whereas VPA might sensitize the cells to etoposide, inappropriate sequence of cultivation of the cells with VPA can decrease the etoposide cytotoxic efficacy. The results found here warrant further studies of combined treatment of neuroblastoma cells with etoposide with HDAC inhibitors and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.

Mechanisms of ellipticine-mediated resistance in UKF-NB-4 neuroblastoma cells.

Most high-risk neuroblastomas develop resistance to cytostatics and therefore there is a need to develop new drugs. In previous studies, we found that ellipticine induces apoptosis in human neuroblastoma cells. We also investigated whether ellipticine was able to induce resistance in the UKF-NB-4 neuroblastoma line and concluded that it may be possible after long-term treatment with increasing concentrations of ellipticine. The aim of the present study was to investigate the mechanisms responsible for ellipticine resistance. To elucidate the mechanisms involved, we used the ellipticine-resistant subline UKF-NB-4(ELLI) and performed comparative genomic hybridization, multicolor and interphase FISH, expression microarray, real-time RT-PCR, flow cytometry and western blotting analysis of proteins. On the basis of our results, it appears that ellipticine resistance in neuroblastoma is caused by a combination of overexpression of Bcl-2, efflux or degradation of the drug and downregulation of topoisomerases. Other mechanisms, such as upregulation of enzymes involved in oxidative phosphorylation, cellular respiration, V-ATPases, aerobic respiration or spermine synthetase, as well as reduced growth rate, may also be involved. Some changes are expressed at the DNA level, including gains, amplifications or deletions. The present study demonstrates that resistance to ellipticine is caused by a combination of mechanisms.

Valproic acid overcomes hypoxia-induced resistance to apoptosis.

Valproic acid (VPA), a histone deacetylase inhibitor (HDACi), has been shown to be an effective tool in cancer treatment. Although its ability to induce apoptosis has been described in many cancer types, the data come from experiments performed in normoxic (21% O2) conditions only. Therefore, we questioned whether VPA would be equally effective under hypoxic conditions (1% O2), which is known to induce resistance to apoptosis. Four neuroblastoma cell lines were used: UKF-NB-3, SK-N-AS, plus one cisplatin-resistant subline derived from each of the two original sensitive lines. All were treated with VPA and incubated under hypoxic conditions. Measurement of apoptosis and viability using TUNEL assay and Annexin V/propidium iodide labeling revealed that VPA was even more effective under hypoxic conditions. We show here that hypoxia-induced resistance to chemotherapeutic agents such as cisplatin could be overcome using VPA. We also demonstrated that apoptosis pathways induced by VPA do not differ between normoxic and hypoxic conditions. VPA-induced apoptosis proceeds through the mitochondrial pathway, not the extrinsic pathway (under both normoxia and hypoxia), since inhibition of caspase-8 failed to decrease apoptosis or influence bid cleavage. Our data demonstrated that VPA is more efficient in triggering apoptosis under hypoxic conditions and overcomes hypoxia-induced resistance to cisplatin. The results provide additional evidence for the use of VPA in neuroblastoma (NBL) treatment.

Anticancer agent ellipticine combined with histone deacetylase inhibitors, valproic acid and trichostatin A, is an effective DNA damage strategy in human neuroblastoma.

OBJECTIVES: Valproic acid (VPA) and trichostatin A (TSA) exert antitumor activity as histone deacetylase inhibitors, whereas ellipticine action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of cytochrome P450 (CYP)- and peroxidase-mediated covalent DNA adducts. This is the first report on the molecular mechanism of combined treatment of human neuroblastoma UKF-NB-3 and UKF-NB-4 cells with these compounds. METHODS: HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites formed by microsomes and peroxidases. Covalent DNA modifications by ellipticine in neuroblastoma cells and in incubations with microsomes and peroxidases were detected by 32P-postlabeling. Expression of CYP enzymes, peroxidases and cytochrome b5 was examined by Western blot. RESULTS: The cytotoxicity of ellipticine to neuroblastomas was increased by pre-treating these cells with VPA or TSA. A higher sensitivity of cells to ellipticine correlated with an increase in formation of covalent ellipticine-derived DNA adducts in these cells. To evaluate the mechanisms of this finding, we investigated the modulation by VPA and TSA of CYP- and peroxidase-mediated ellipticine-derived DNA adduct formation in vitro. The effects of ellipticine in the presence of VPA and TSA on expression of CYPs and peroxidases relevant for ellipticine activation and levels of cytochrome b5 and P-glycoprotein in neuroblastoma cells were also investigated. Based on these studies, we attribute most of the enhancing effects of VPA and TSA on ellipticine cytotoxicity to enhanced ellipticine-DNA adduct formation caused by an increase in levels of cytochrome b5, CYP3A4 and CYP1A1 in neuroblastoma cells. A lower sensitivity of UKF-NB-4 cells to combined effects of ellipticine with VPA and TSA than of UKF-NB-3 cells is also attributable to high levels of P-glycoprotein expressed in this cell line. CONCLUSION: The results found here warrant further studies and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.

Ellipticine cytotoxicity to cancer cell lines - a comparative study.

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.

DNA and histone deacetylases as targets for neuroblastoma treatment.

Neuroblastoma, a tumor of the peripheral sympathetic nervous system, is the most frequent solid extra cranial tumor in children and is a major cause of death from neoplasia in infancy. Still little improvement in therapeutic options has been made, requiring a need for the development of new therapies. In our laboratory, we address still unsettled questions, which of mechanisms of action of DNA-damaging drugs both currently use for treatment of human neuroblastomas (doxorubicin, cis-platin, cyclophosphamide and etoposide) and another anticancer agent decreasing growth of neuroblastomas in vitro, ellipticine, are predominant mechanism(s) responsible for their antitumor action in neuroblastoma cell lines in vitro. Because hypoxia frequently occurs in tumors and strongly correlates with advanced disease and poor outcome caused by chemoresistance, the effects of hypoxia on efficiencies and mechanisms of actions of these drugs in neuroblastomas are also investigated. Since the epigenetic structure of DNA and its lesions play a role in the origin of human neuroblastomas, pharmaceutical manipulation of the epigenome may offer other treatment options also for neuroblastomas. Therefore, the effects of histone deacetylase inhibitors on growth of neuroblastoma and combination of these compounds with doxorubicin, cis-platin, etoposide and ellipticine as well as mechanisms of such effects in human neuroblastona cell lines in vitro are also investigated. Such a study will increase our knowledge to explain the proper function of these drugs on the molecular level, which should be utilized for the development of new therapies for neuroblastomas.

Isolation and partial characterization of catechol 1,2-dioxygenase of phenol degrading yeast Candida tropicalis.

OBJECTIVES: Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole carbon and energy source. While the enzyme participating on the first step of phenol biodegradation, NADPH-dependent phenol hydroxylase, has already been characterized, information on the enzyme participating in the second step of its degradation, catechol 1,2-dioxygenase, is scarce. The development of the procedure suitable for catechol 1,2-dioxygenase isolation and partial characterization of this enzyme are the aims of this study. METHODS: Combination of chromatography on DEAE-Sepharose and gel-permeation chromatography on Sephadex G-100 was used for isolation of cytosolic catechol 1,2-dioxygenase from C. tropicalis yeast. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephadex G-100 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation). RESULTS: Using the isolation procedure consisting of chromatography and re-chromatography on a column of DEAE-Sepharose and gel filtration on Sephadex G-100, catechol 1,2-dioxygenase was purified from C. tropicalis cytosol to homogeneity. Catechol 1,2-dioxygenase was found to be a homodimer with a subunit molecular mass of 30000 +/- 5000. The enzyme oxidized catechol producing cis,cis-muconic acid. The optimal temperature and pH were 30 degrees C and 7.7, respectively. CONCLUSIONS: The data are the first report showing the isolation of eukaryotic catechol 1,2-dioxygenase from C. tropicalis to homogeneity and its partial characterization.

The mechanism of cytotoxicity and DNA adduct formation by the anticancer drug ellipticine in human neuroblastoma cells.

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 inhibitors valproate and trichostatin A are toxic to neuroblastoma cells and modulate cytochrome P450 1A1, 1B1 and 3A4 expression in these cells.

Histone deacetylase inhibitors such as valproic acid (VPA) and trichostatin A (TSA) were shown to exert antitumor activity. Here, the toxicity of both drugs to human neuroblastoma cell lines was investigated using MTT test, and IC50 values for both compounds were determined. Another target of this work was to evaluate the effects of both drugs on expression of cytochrome P450 (CYP) 1A1, 1B1 and 3A4 enzymes, which are known to be expressed in neuroblastoma cells. A malignant subset of neuroblastoma cells, so-called N-type cells (UKF-NB-3 cells) and the more benign S-type neuroblastoma cells (UKF-NB-4 and SK-N-AS cell lines) were studied from both two points of view. VPA and TSA inhibited the growth of neuroblastoma cells in a dose-dependent manner. The IC(50) values ranging from 1.0 to 2.8 mM and from 69.8 to 129.4 nM were found for VPA and TSA, respectively. Of the neuroblastoma tested here, the N-type UKF-NB-3 cell line was the most sensitive to both drugs. The different effects of VPA and TSA were found on expression of CYP1A1, 1B1 and 3A4 enzymes in individual neuroblastoma cells tested in the study. Protein expression of all these CYP enzymes in the S-type SK-N-AS cell line was not influenced by either of studied drugs. On the contrary, in another S-type cell line, UKF-NB-4, VPA and TSA induced expression of CYP1A1, depressed levels of CYP1B1 and had no effect on expression levels of CYP3A4 enzyme. In the N-type UKF-NB-3 cell line, the expression of CYP1A1 was strongly induced, while that of CYP1B1 depressed by VPA and TSA. VPA also induced the expression of CYP3A4 in this neuroblastoma cell line.

Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2.

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).

The comparison of cytotoxicity of the anticancer drugs doxorubicin and ellipticine to human neuroblastoma cells.

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 cytotoxicity of ellipticine to human neuroblastoma derived cell lines IMR-32 and UKF-NB-4 was investigated. Treatment of neuroblastoma cells with ellipticine was compared with that of these cancer cells with doxorubicin. The toxicity of ellipticine was essentially the same as that of doxorubicin to UKF-NB-4 cells, but doxorubicin is much more effective to inhibit the growth of the IMR-32 cell line than ellipticine. Hypoxic conditions used for the cell cultivation resulted in a decrease in ellipticine and/or doxorubicin toxicity to IMR-32 and UKF-NB-4 neuroblastoma cells.

DNA adduct formation by the anticancer drug ellipticine in human leukemia HL-60 and CCRF-CEM cells.

Ellipticine induces formation of two DNA adducts in leukemia HL-60 and CCRF-CEM cells, identical with deoxyguanosine adducts generated by ellipticine metabolites 13-hydroxyellipticine and 12-hydroxyellipticine in vitro and in vivo. The ellipticine cytotoxicity to HL-60 (IC(50)=0.64microM) and CCRF-CEM cells (IC(50)=4.7microM) correlates with levels of DNA adducts. The different expressions of enzymes activating ellipticine in cells explain this finding. While cytochrome P450 1A1 and cyclooxygenase-1 are expressed in both cells, HL-60 cells express also high levels of another activator, myeloperoxidase. The results suggest the adduct formation as a new mode of antitumor action of ellipticine for leukemia.

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.

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.

Cytochromes P450 reconstituted with NADPH: P450 reductase mimic the activating and detoxicating metabolism of the anticancer drug ellipticine in microsomes.

OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple action mechanisms. Recently, we found that after cytochrome P450 (CYP)-mediated oxidation ellipticine forms covalent DNA adducts. Ellipticine oxidation by isolated CYP and its binding to DNA is the target of this study. METHODS: High performance liquid chromatography (HPLC) was employed for separation and characterization of ellipticine metabolites generated by CYPs. The (32)P-postlabeling technique was utilized to determine ellipticine-DNA adducts. RESULTS: Purified CYP enzymes reconstituted with NADPH:CYP reductase oxidized ellipticine to up to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only CYP1A1 was capable to form all metabolites. Using the reconstituted enzymatic system, we demonstrated that the detoxication ellipticine metabolites, 7-hydroxyellipticine and 9-hydroxyellipticine, are mainly generated by CYP1A1 and 1A2, while those responsible for DNA binding, 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, by CYP3A1 and 2C3. Likewise, the most efficient CYPs forming DNA adducts from ellipticine were CYP3A1 and 2C3. CONCLUSIONS: The results showed that the system of purified CYPs reconstituted with NADPH: CYP reductase proved for ellipticine oxidation provide a true reflection of the situation in the microsomal membrane.

Oxidation of an antitumor drug ellipticine by peroxidases.

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Since we found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts, this anticancer drug is considered to function as a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its enzymatic activation in target tissues. Here, we demonstrate that ellipticine is also oxidized by peroxidases, which are abundantly expressed in several target tumor tissues. Lactoperoxidase, myeloperoxidase and horseradish peroxidase were used as models. Peroxidases in the presence of hydrogen peroxide oxidize ellipticine to an ellipticine dimer and N(2)-oxide of ellipticine as the major and minor metabolite, respectively. Inhibition of the peroxidase-mediated ellipticine oxidation by radical scavengers ascorbate, glutathione and NADH suggests a one-electron mechanism of the oxidation. The implication of the oxidation of ellipticine by peroxidases in its mechanism of action is discussed.

The anticancer drug ellipticine forms covalent DNA adducts, mediated by human cytochromes P450, through metabolism to 13-hydroxyellipticine and ellipticine N2-oxide.

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.