Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
ReviewCytochrome P450- and peroxidase-mediated oxidation of anticancer alkaloid ellipticine dictates its anti-tumor efficiency☆
Introduction
Cytochromes P450 (CYP)1 are a superfamily of heme proteins distributed widely throughout nature, involved in metabolism of a broad variety of substrates and catalyzing a variety of interesting chemical reactions. They play a central role in metabolism of chemotherapeutic agents [1], [2]. Many well-defined examples exist for roles of CYPs in decreasing the adverse effects of drugs through biotransformation, and an equally interesting field of investigation is the bioactivation of chemicals, including drugs. Several prodrug anti-tumor agents have been found as CYP substrates for which tumor CYP activation was identified. Those in clinical use include prodrug alkylating agents such as cyclophosphamide, ifosphamide, dacarbazine and procarbazine, a metabolically activated anthracycline, as well as tamoxifen [3]. These prodrugs were found to be significantly more active following CYP-hydroxylation [3]. A wide range of human cancers is known to express the drug metabolizing CYPs, notably 1A, 1B, 2C, 3A and 2D subfamily members [4]. Individual tumor types have distinct CYP profiles as studied by detection of CYP activity, identification of immunoreactive CYP protein and detection of CYP mRNA [3]. This raises the possibility that tumor CYP enzymes could be a focus for tumor-specific prodrug activation.
Besides CYPs, a variety of xenobiotics such as drugs and carcinogens are also metabolized by peroxidases. Lactoperoxidase (LPO), myeloperoxidase (MPO), cyclooxygenase (COX)-1, and COX-2 are expressed in multiple cancer types such as myeloblastic leukemia [5], [6], carcinomas, brain tumors [7] and some pre-neoplastic lesions. One of them, COX-2, is even inducible by carcinogenic processes and/or by several compounds, including anticancer drugs [8], [9], [10], [11], [12], [13]. During the peroxidase-mediated oxidation of xenobiotics, they are not only detoxified, but also activated to reactive species binding covalently to macromolecules such as DNA [14], [15], [16]. Such processes might be implicated both in cancer initiation and in its chemotherapy.
Therefore, the enzymes metabolizing xenobiotic may play an essential role in modulation of efficiencies of various anticancer drugs in target tissues, and the expression of these enzymes is crucial for such effects. This review summarizes a role of CYP and peroxidase enzymes in anti-tumor efficiency of an anticancer plant alkaloid ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole, NSC 69178, Fig. 1).
Ellipticine, an alkaloid found in Apocyanaceae plants (i.e. Ochrosia borbonica, Excavatia coccinea), is one of the simplest naturally occurring alkaloids, having a planar structure [17]. It was first isolated in 1959 from the leaves of the evergreen tree Ochrosia elliptica, which grows wild in Oceania [17]. In preclinical experiments and in clinical trials, this compound and several of its more soluble derivatives (9-hydroxyellipticine, 9-hydroxy-N2-methylellipticinium, 9-chloro-N2-methylellipticinium, 9-methoxy-N2-methylellipticinium, and 9-dimethyl-amino-ethoxy-ellipticine) exhibit significant anti-tumor and anti-HIV activities [18], [19]. They showed activity against cancer cell lines such as leukemias (L1210, P388, HL-60, and CCRF-CEM cell lines), lymphosarcomas, B16 melanoma, colon cancer SW480 cell line, Lewis lung carcinoma, human non-small-cell-lung-cancer, hepatocellular carcinoma HepG2 cells, glioblastoma U87MG and U373 cells, osteosarcoma, breast adenocarcinoma MCF-7 cells and neuroblastoma cells (IMR-32, UKF-NB-3, and UKF-NB-4 cell lines), which are killed at concentrations of ellipticine and its derivatives ranging from 10−10 to 10−6 (for an overview see literature [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37]). One of the ellipticine derivatives, 9-hydroxy-N2-methylellipticine (NSC 264-137), seems particularly improving the condition of patients suffering from breast cancer [38] and osteolytic breast cancer metastasis [18], [39], [40], [41], [42]. Activity against anaplastic thyroid carcinoma and ovarian carcinoma has also been observed. Most patients usually received a weekly perfusion of 80 mg/m2 [39], [40].
The main reason for the interest in ellipticine and its derivatives for clinical purposes is their high efficiencies against several types of cancer and their complete lack of hematologic and hepatic toxicity [20]. In patients which received a weekly perfusion of 80 mg/m2 of 9-hydroxy-N2-methylellipticine, no renal trouble was observed during the first year, but 2 deaths from renal insufficiency occurred during the 18th and 15th months of treatment. Nevertheless, the most frequent adverse effect consists of digestive troubles (nausea and vomiting in one-third of the patients) which rarely compelled stopping the treatment (4 times in 100 patients), hypertension (less than 10% of the patients), muscular cramp (one-third of the patients), fatigue which can be very pronounced (in most patients after 3 months of treatment), mouth dryness, and mycosis of the tongue and esophagus (less than 20% of the patients) [39], [40]. In addition, mutagenicity of ellipticines should be evaluated as a potential risk factor for these anticancer agents. Namely, 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 [27]). In addition, ellipticine was found to give positive results in mouse lymphoma assay [43], in the forward gene mutation at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells [44] and in the induction of sister-chromatid exchanges in cultured mammalian cells [45], [46].
The anti-tumor therapeutic ellipticine and its derivatives act as potent anti-tumor agents via a combined mechanism of cell cycle arrest and induction of the apoptotic pathway. Ellipticine has been reported (i) to arrest cell cycle progression by regulating the expression of cyclinB1 and Cdc2 as well as phosphorylation of Cdc2 in human breast cancer cell lines [47], [48], (ii) to induce apoptotic cell death by the generation of cytotoxic free radicals, the activation of Fas/Fas ligand system and the regulation of Bcl-2 family proteins in human breast cancer and hepatocellular carcinoma HepG2 cells [31], [47], [48], [49], and (iii) to cause the initiation of the mitochondrial apoptosis pathway [30], [31], [47], [48].
Ellipticine also uncouples mitochondrial oxidative phosphorylation [50] and thereby disrupts the energy balance of cells. In the case of ellipticine derivative 9-dimethyl-amino-ethoxy-ellipticine (NSC 338258, EPED3) in myeloma cells, NSC 338258-induced cell cycle arrest and an apoptotic progression that appears to be a consequence of the instantaneous effect of the drug on cytoplasmic organelles, particularly mitochondria. Disruption of mitochondria and cytoplasmic distribution of cytochrome c initiated the intracellular proteolytic cascade through the intrinsic apoptotic pathway. Moreover, NSC 338258 is able to induce apoptosis in myeloma cells with de novo or acquired resistance to commonly administered antimyeloma agents [34]. Studies on the mechanisms of the cytotoxic and anticancer activity of ellipticines have also shown that these activities might be due to induction of endoplasmic reticulum stress [51].
Numerous studies have reported the involvement of p53 tumor suppressor protein in ellipticine cytotoxic effects [24], [26], [37], [48], [52], [53]. 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 [52]. 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 [24], and this correlated with their cytotoxic activity. Moreover, accumulation of dephosphorylated mutant p53 might induce apoptosis [24]. Ellipticine has also been found to restore the transcription function of mutant p53. This property may contribute to the selectivity of ellipticine-derived compounds against tumor cell lines expressing mutant p53 [53]. In human breast adenocarcinoma MCF-7 cells ellipticine causes G2/M phase arrest of the cell cycle associated with an increase in the protein expression of p53, p21/WAF1, and KIP1/p27, and growth inhibition by this compound is also induced by mitochondrial proapoptotic pathways in these cells [48]. Ellipticine might also function as a modulator of p53 nuclear localization; in HCT116 colon cancer cells ellipticine increased the nuclear localization of endogenous p53 with a resultant increase in the transactivation of the p21 promoter [54]. Recently, we have found that ellipticine also activates the p53 pathway in glioblastoma cells; its impact on these cancer cells depends on the p53 status. In a U87MG glioblastoma cell line expressing p53wt, ellipticine provoked an early G0/G1 cell cycle arrest, whereas in a U373 cell line expressing p53mt it caused arrest in S and G2/M phases [37].
However, the precise molecular mechanism responsible for these effects has not yet been explained. Chemotherapy-induced cell cycle arrest was shown to result from DNA damages caused by a variety of chemotherapeutics. In the case of ellipticine, it was suggested that the prevalent DNA-mediated mechanisms of their anti-tumor, mutagenic and cytotoxic activities are (i) intercalation into DNA [20], [55] and (ii) inhibition of DNA topoisomerase II activity [20], [56], [57], [58]. 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 [55], [59].
The mechanism of ellipticine action as the inhibitor of topoisomerase II has also been extensively studied [56], [57], [58]. 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 [58] 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.
It is evident that the explanations of anticancer activity mentioned above are based on mechanisms of nonspecific drug actions. Intercalation of ellipticine into DNA and inhibition of topoisomerase II occur in all cell types irrespective of their metabolic capacity, because of the general chemical properties of this drug and its affinity to DNA and topoisomerase II protein [20], [28], [30]. In addition, other ellipticine effects described above, and the transport of highly hydrophobic ellipticine molecules across cell membranes into cells (including both tumor and healthy cells), are nonspecific. However, this contrasts with specificity of antineoplastic activity of ellipticines against only several types of cancer diseases. The specificity of anti-tumor activity of ellipticines should, hence, be a consequence of other mechanisms of their action, which have not been elucidated in the former studies.
Recently, we have confirmed this suggestion. We have demonstrated that ellipticine also covalently binds to DNA in vitro and in vivo after being enzymatically activated with CYP and/or peroxidase enzymes [27], [28], [32], [33], [60], [61], [62], [63], [64], [65], [66], [67], suggesting a third mechanism of action based on DNA damage. Using [3H]elipticine and 32P-postlabeling method, we have found that during the ellipticine oxidation by CYPs and peroxidases two major and several minor ellipticine-derived adducts are generated in DNA in vitro and in rat and mouse models in vivo (Fig. 2A–P) [27], [60], [61], [62], [63], [64], [65], [66], [67].
Section snippets
Oxidation of ellipticine by cytochromes P450 plays a role both in its activation to species binding to DNA and in its detoxication
A variety of human, rat, rabbit, and mouse CYP enzymes oxidize ellipticine, forming up to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine, and ellipticine N2-oxide (Fig. 1) [62], [65], [68]. Of human CYP enzymes, CYP1A1, 1A2, and 1B1 are the major enzymes oxidizing ellipticine to 9-hydroxy- and 7-hydroxyellipticine (Fig. 1, Fig. 3). Since these metabolites are excreted from organisms, they are considered to be mainly the detoxication products of ellipticine
Oxidation of ellipticine by peroxidases is another pathway leading to metabolic activation and detoxication of ellipticine
While CYP enzymes are abundantly expressed in several healthy tissues, in which the DNA adducts were detected in vivo [60], [64], [65], and in some target cancers like breast cancer [4], [83], [84], [85], 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 and the T-cell leukemia CCRF-CEM cells and generates the DNA adducts in these cells (Fig. 2M, N) [32], even
Conclusions
The data summarized in this review demonstrate that an anticancer alkaloid ellipticine, which acts as DNA-damaging agent, might be considered a prodrug, whose anti-tumor efficiency and/or genotoxic side effects are dependent on its activation by CYPs and peroxidases in target tissues. These activation enzymes, when expressed in tumor cells, confer the ability to metabolize this anticancer prodrug into more potent cytotoxins. Two of such metabolites, 13-hydroxyellipticine and
Acknowledgments
The work was supported by the Grant Agency of the Czech Republic (grant P301/10/0356), and the Ministry of Education of the Czech Republic (grants MSM0021620808 and 1M0505). We thank Dr. Norbert Frank (German Cancer Research Center, Heidelberg) for providing female rats bearing breast adenocarcinoma.
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2019, Toxicology and Applied PharmacologyCitation Excerpt :Ellipticine (5,11-dimethyl-6H-pyrido[4,5-b]carbazole) is a cytotoxic alkaloid isolated from the Apocynaceae family of plants. Both ellipticine and its derivatives possess anti-HIV and anti-tumour properties allowing it to be used against several cancers with limited toxic side effects and no haematological toxicity by functioning through multiple mechanisms that result in cell cycle arrest and initiation of apoptosis (Martinkova et al., 2010; Stiborova et al., 2011; Miller and McCarthy, 2012; Stiborova and Frei, 2014). The main mechanisms by which ellipticine exerts its anti-tumour, cytotoxic and mutagenic effects are inhibition of topoisomerase II, intercalation into DNA and enzyme-mediated formation of covalent ellipticine-derived DNA adducts (Garbett and Graves, 2004; Stiborova and Frei, 2014; Banerjee et al., 2015; Vann et al., 2016).
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Dedicated to Professor Klaus Ruckpaul on occasion of his 80th birthday.