PURPOSE: The chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential. METHODS: The Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed. RESULTS: Ex ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM. CONCLUSIONS: Taken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

• Klíčová slova
• CAM assay, drug testing, extracranial solid tumor, metastasis, neuroblastoma, preclinical trials,
• Publikační typ
• časopisecké články MeSH

Herein, the in vitro metabolism of tyrosine kinase inhibitor cabozantinib, the drug used for the treatment of metastatic medullary thyroid cancer and advanced renal cell carcinoma, was studied using hepatic microsomal samples of different human donors, human recombinant cytochromes P450 (CYPs), flavin-containing mono-oxygenases (FMOs) and aldehyde oxidase. After incubation with human microsomes, three metabolites, namely cabozantinib N-oxide, desmethyl cabozantinib and monohydroxy cabozantinib, were detected. Significant correlations were found between CYP3A4 activity and generation of all metabolites. The privileged role of CYP3A4 was further confirmed by examining the effect of CYP inhibitors and by human recombinant enzymes. Only four of all tested human recombinant cytochrome P450 were able to oxidize cabozantinib, and CYP3A4 exhibited the most efficient activity. Importantly, cytochrome b5 (cyt b5) stimulates the CYP3A4-catalyzed formation of cabozantinib metabolites. In addition, cyt b5 also stimulates the activity of CYP3A5, whereas two other enzymes, CYP1A1 and 1B1, were not affected by cyt b5. Since CYP3A4 exhibits high expression in the human liver and was found to be the most efficient enzyme in cabozantinib oxidation, we examined the kinetics of this oxidation. The present study provides substantial insights into the metabolism of cabozantinib and brings novel findings related to cabozantinib pharmacokinetics towards possible utilization in personalized medicine.

• Klíčová slova
• cabozantinib, cytochrome P450, cytochrome b5, tyrosine kinase inhibitor,
• Publikační typ
• časopisecké články MeSH

The anticancer drug ellipticine exerts its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. The present study has examined the role of cytochrome P450 oxidoreductase (POR) and cytochrome b5 (Cyb5), electron donors to P450 enzymes, in the CYP-mediated metabolism and disposition of ellipticine in vivo. We used Hepatic Reductase Null (HRN) and Hepatic Cytochrome b5/P450 Reductase Null (HBRN) mice. HRN mice have POR deleted specifically in hepatocytes; HBRN mice also have Cyb5 deleted in the liver. Mice were treated once with 10 mg/kg body weight ellipticine (n = 4/group) for 24 h. Ellipticine-DNA adduct levels measured by 32P-postlabelling were significantly lower in HRN and HBRN livers than in wild-type (WT) livers; however no significant difference was observed between HRN and HBRN livers. Ellipticine-DNA adduct formation in WT, HRN and HBRN livers correlated with Cyp1a and Cyp3a enzyme activities measured in hepatic microsomes in the presence of NADPH confirming the importance of P450 enzymes in the bioactivation of ellipticine in vivo. Hepatic microsomal fractions were also utilised in incubations with ellipticine and DNA in the presence of NADPH, cofactor for POR, and NADH, cofactor for Cyb5 reductase (Cyb5R), to examine ellipticine-DNA adduct formation. With NADPH adduct formation decreased as electron donors were lost which correlated with the formation of the reactive metabolites 12- and 13-hydroxy-ellipticine in hepatic microsomes. No difference in adduct formation was observed in the presence of NADH. Our study demonstrates that Cyb5 contributes to the P450-mediated bioactivation of ellipticine in vitro, but not in vivo.

• Klíčová slova
• Cytochrome P450, Cytochrome b(5), DNA Adducts, Metabolism, Mouse models,
• MeSH
• adukty DNA metabolismus   MeSH
• aromatické hydroxylasy metabolismus   MeSH
• cytochrom P-450 CYP3A MeSH
• cytochrom-B(5)-reduktasa nedostatek   genetika   MeSH
• cytochromy b5 nedostatek   genetika   MeSH
• elipticiny metabolismus   farmakologie   MeSH
• fenotyp MeSH
• genotyp MeSH
• hepatocyty enzymologie   MeSH
• jaterní mikrozomy enzymologie   MeSH
• játra enzymologie   MeSH
• metabolická aktivace MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• NADPH-cytochrom c-reduktasa metabolismus   MeSH
• protinádorové látky metabolismus   farmakologie   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• aromatické hydroxylasy MeSH
• CYP3A protein, mouse MeSH Prohlížeč
• cytochrom P-450 CYP3A MeSH
• cytochrom-B(5)-reduktasa MeSH
• cytochromy b5 MeSH
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• NADPH-cytochrom c-reduktasa MeSH
• protinádorové látky MeSH
• systém (enzymů) cytochromů P-450 MeSH

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-) or TP53(-/-) were treated for up to 48 h with 60 μM cisplatin, 50 μM etoposide or 5 μM ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60-80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(-/-) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5 μM BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(-/-) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.

• Klíčová slova
• Benzo[a]pyrene, Cisplatin, Cytochrome P450, Ellipticine, Etoposide, Tumour suppressor p53,
• MeSH
• adukty DNA metabolismus   MeSH
• benzopyren farmakokinetika   farmakologie   MeSH
• cisplatina farmakologie   MeSH
• cytochrom P-450 CYP1A1 biosyntéza   metabolismus   MeSH
• cytochrom P-450 CYP3A biosyntéza   metabolismus   MeSH
• elipticiny farmakokinetika   farmakologie   MeSH
• enzymová indukce účinky léků   MeSH
• etoposid farmakologie   MeSH
• geny p53 MeSH
• HCT116 buňky MeSH
• karcinogeny farmakokinetika   farmakologie   MeSH
• kolorektální nádory farmakoterapie   genetika   metabolismus   patologie   MeSH
• lidé MeSH
• metabolická aktivace MeSH
• nádorový supresorový protein p53 nedostatek   genetika   metabolismus   MeSH
• poškození DNA MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• benzopyren MeSH
• cisplatina MeSH
• CYP1A1 protein, human MeSH Prohlížeč
• CYP3A4 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP1A1 MeSH
• cytochrom P-450 CYP3A MeSH
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• etoposid MeSH
• karcinogeny MeSH
• nádorový supresorový protein p53 MeSH
• TP53 protein, human MeSH Prohlížeč

Neuroblastoma (NBL) originates from undifferentiated cells of the sympathetic nervous system. Chemotherapy is judged to be suitable for successful treatment of this disease. Here, the influence of histone deacetylase (HDAC) inhibitor valproate (VPA) combined with DNA-damaging chemotherapeutic, ellipticine, on UKF-NB-4 and SH-SY5Y neuroblastoma cells was investigated. Treatment of these cells with ellipticine in combination with VPA led to the synergism of their anticancer efficacy. The effect is more pronounced in the UKF-NB-4 cell line, the line with N-myc amplification, than in SH-SY5Y cells. This was associated with caspase-3-dependent induction of apoptosis in UKF-NB-4 cells. The increase in cytotoxicity of ellipticine in UKF-NB-4 by VPA is dictated by the sequence of drug administration; the increased cytotoxicity was seen only after either simultaneous exposure to these drugs or after pretreatment of cells with ellipticine before their treatment with VPA. The synergism of treatment of cells with VPA and ellipticine seems to be connected with increased acetylation of histones H3 and H4. Further, co-treatment of cells with ellipticine and VPA increased the formation of ellipticine-derived DNA adducts, which indicates an easier accessibility of ellipticine to DNA in cells by its co-treatment with VPA and also resulted in higher ellipticine cytotoxicity. The results are promising for in vivo studies and perhaps later for clinical studies of combined treatment of children suffering from high-risk NBL.

• Klíčová slova
• DNA damage, acetylation of histones, apoptosis, ellipticine, neuroblastoma, valproate,
• MeSH
• apoptóza MeSH
• elipticiny toxicita   MeSH
• inhibitory histondeacetylas toxicita   MeSH
• kyselina valproová toxicita   MeSH
• lidé MeSH
• mutageny toxicita   MeSH
• nádorové buněčné linie MeSH
• neuroblastom metabolismus   MeSH
• neurony účinky léků   metabolismus   MeSH
• synergismus léků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• inhibitory histondeacetylas MeSH
• kyselina valproová MeSH
• mutageny MeSH

ABSTRACT: Ellipticine is an anticancer agent that forms covalent DNA adducts after enzymatic activation by cytochrome P450 (CYP) enzymes, mainly by CYP3A4. This process is one of the most important ellipticine DNA-damaging mechanisms for its antitumor action. Here, we investigated the efficiencies of human hepatic microsomes and human recombinant CYP3A4 expressed with its reductase, NADPH:CYP oxidoreductase (POR), NADH:cytochrome b5 reductase and/or cytochrome b5 in Supersomes™ to oxidize this drug. We also evaluated the effectiveness of coenzymes of two of the microsomal reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of NADH:cytochrome b5 reductase, to mediate ellipticine oxidation in these enzyme systems. Using HPLC analysis we detected up to five ellipticine metabolites, which were formed by human hepatic microsomes and human CYP3A4 in the presence of NADPH or NADH. Among ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine were formed by hepatic microsomes as the major metabolites, while 7-hydroxyellipticine and the ellipticine N2-oxide were the minor ones. Human CYP3A4 in Supersomes™ generated only three metabolic products, 9-hydroxy-, 12-hydroxy-, and 13-hydroxyellipticine. Using the 32P-postlabeling method two ellipticine-derived DNA adducts were generated by microsomes and the CYP3A4-Supersome system, both in the presence of NADPH and NADH. These adducts were derived from the reaction of 13-hydroxy- and 12-hydroxyellipticine with deoxyguanosine in DNA. In the presence of NADPH or NADH, cytochrome b5 stimulated the CYP3A4-mediated oxidation of ellipticine, but the stimulation effect differed for individual ellipticine metabolites. This heme protein also stimulated the formation of both ellipticine-DNA adducts. The results demonstrate that cytochrome b5 plays a dual role in the CYP3A4-catalyzed oxidation of ellipticine: (1) cytochrome b5 mediates CYP3A4 catalytic activities by donating the first and second electron to this enzyme in its catalytic cycle, indicating that NADH:cytochrome b5 reductase can substitute NADPH-dependent POR in this enzymatic reaction and (2) cytochrome b5 can act as an allosteric modifier of the CYP3A4 oxygenase.

• Klíčová slova
• Coenzymes, DNA, Enzymes, High pressure liquid chromatography,
• Publikační typ
• časopisecké články MeSH

Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after activation by cytochrome P450 (P450). Here, we investigated whether NADH:cytochrome b5 reductase (CBR) in the presence of cytochrome b5 can act as sole electron donor to human P450 1A1 during BaP oxidation and replace the canonical NADPH:cytochrome P450 reductase (POR) system. We also studied the efficiencies of the coenzymes of these reductases, NADPH as a coenzyme of POR, and NADH as a coenzyme of CBR, to mediate BaP oxidation. Two systems containing human P450 1A1 were utilized: human recombinant P450 1A1 expressed with POR, CBR, epoxide hydrolase, and cytochrome b5 in Supersomes and human recombinant P450 1A1 reconstituted with POR and/or with CBR and cytochrome b5 in liposomes. BaP-9,10-dihydrodiol, BaP-7,8-dihydrodiol, BaP-1,6-dione, BaP-3,6-dione, BaP-9-ol, BaP-3-ol, a metabolite of unknown structure, and two BaP-DNA adducts were generated by the P450 1A1-Supersomes system, both in the presence of NADPH and in the presence of NADH. The major BaP-DNA adduct detected by (32)P-postlabeling was characterized as 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-BaP (assigned adduct 1), while the minor adduct is probably a guanine adduct derived from 9-hydroxy-BaP-4,5-epoxide (assigned adduct 2). BaP-3-ol as the major metabolite, BaP-9-ol, BaP-1,6-dione, BaP-3,6-dione, an unknown metabolite, and adduct 2 were observed in the system using P450 1A1 reconstituted with POR plus NADPH. When P450 1A1 was reconstituted with CBR and cytochrome b5 plus NADH, BaP-3-ol was the predominant metabolite too, and an adduct 2 was also generated. Our results demonstrate that the NADH/cytochrome b5/CBR system can act as the sole electron donor both for the first and second reduction of P450 1A1 during the oxidation of BaP in vitro. They suggest that NADH-dependent CBR can replace NADPH-dependent POR in the P450 1A1-catalyzed metabolism of BaP.

• MeSH
• adukty DNA metabolismus   MeSH
• benzopyren toxicita   MeSH
• cytochrom-B(5)-reduktasa metabolismus   MeSH
• lidé MeSH
• oxidace-redukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• benzopyren MeSH
• cytochrom-B(5)-reduktasa MeSH

ABSTRACT: Cytochrome P450 (CYP) 2S1 is "orphan" CYP that is overexpressed in several epithelial tissues and many human tumors. The pure enzyme is required for better understanding of its biological functions. Therefore, human CYP2S1 was considered to be prepared by the gene manipulations and heterologous expression in Escherichia coli. Here, the conditions suitable for efficient expression of human CYP2S1 protein from plasmid pCW containing the human CYP2S1 gene were optimized and the enzyme purified to homogeneity. The identity of CYP2S1 as the product of heterologous expression was confirmed by dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. To confirm the presence of the enzymatically active CYP2S1, the CO spectrum of purified CYP2S1 was recorded. Since CYP2S1 was shown to catalyze oxidation of compounds having polycyclic aromatic structures, the prepared enzyme has been tested to metabolize the compounds having this structural character; namely, the human carcinogen benzo[a]pyrene (BaP), its 7,8-dihydrodiol derivative, and an anticancer drug ellipticine. Reaction mixtures contained besides the test compounds the CYP2S1 enzyme reconstituted with NADPH:CYP reductase (POR) in liposomes, and/or this CYP in the presence of cumene hydroperoxide or hydrogen peroxide. High performance liquid chromatography was employed for separation of BaP, BaP-7,8-dihydrodiol, and ellipticine metabolites. The results found in this study demonstrate that CYP2S1 in the presence of cumene hydroperoxide or hydrogen peroxide catalyzes oxidation of two of the test xenobiotics, a metabolite of BaP, BaP-7,8-dihydrodiol, and ellipticine. Whereas BaP-7,8,9,10-tetrahydrotetrol was formed as a product of BaP-7,8-dihydrodiol oxidation, ellipticine was oxidized to 12-hydroxyellipticine, 13-hydroxyellipticine, and the ellipticine N2-oxide.

• Klíčová slova
• Coenzymes, Enzymes, High pressure liquid chromatography,
• Publikační typ
• časopisecké články MeSH

ABSTRACT: The microsomal protein cytochrome b5 , which is located in the membrane of the endoplasmic reticulum, has been shown to modulate many reactions catalyzed by cytochrome P450 (CYP) enzymes. We investigated the influence of exposure to the anticancer drug ellipticine and to two environmental carcinogens, benzo[a]pyrene (BaP) and 1-phenylazo-2-naphthol (Sudan I), on the expression of cytochrome b5 in livers of rats, both at the mRNA and protein levels. We also studied the effects of these compounds on their own metabolism and the formation of DNA adducts generated by their activation metabolite(s) in vitro. The relative amounts of cytochrome b5 mRNA, measured by real-time polymerase chain reaction analysis, were induced by the test compounds up to 11.7-fold in rat livers. Western blotting using antibodies raised against cytochrome b5 showed that protein expression was induced by up to sevenfold in livers of treated rats. Microsomes isolated from livers of exposed rats catalyzed the oxidation of ellipticine, BaP, and Sudan I and the formation of DNA adducts generated by their reactive metabolite(s) more effectively than hepatic microsomes isolated from control rats. All test compounds are known to induce CYP1A1. This induction is one of the reasons responsible for increased oxidation of these xenobiotics by microsomes. However, induction of cytochrome b5 can also contribute to their enhanced metabolism.

• Klíčová slova
• DNA, Drug research, Enzymes, High pressure liquid chromatography,
• Publikační typ
• časopisecké články MeSH

Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.

• MeSH
• cytochrom P-450 CYP1A1 nedostatek   genetika   metabolismus   MeSH
• elipticiny farmakologie   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• myši MeSH
• poškození DNA * MeSH
• protinádorové látky farmakologie   MeSH
• rozpřahující látky farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• cytochrom P-450 CYP1A1 MeSH
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• protinádorové látky MeSH
• rozpřahující látky MeSH