BACKGROUND: Iron oxide nanoparticles (IONs) have been increasingly utilized in a wide spectrum of biomedical applications. Surface coatings of IONs can bestow a number of exceptional properties, including enhanced stability of IONs, increased loading of drugs or their controlled release. METHODS: Using two-step sonochemical protocol, IONs were surface-coated with polyoxyethylene stearate, polyvinylpyrrolidone or chitosan for a loading of two distinct topo II poisons (doxorubicin and ellipticine). The cytotoxic behavior was tested in vitro against breast cancer (MDA-MB-231) and healthy epithelial cells (HEK-293 and HBL-100). In addition, biocompatibility studies (hemotoxicity, protein corona formation, binding of third complement component) were performed. RESULTS: Notably, despite surface-coated IONs exhibited only negligible cytotoxicity, upon tethering with topo II poisons, synergistic or additional enhancement of cytotoxicity was found in MDA-MB-231 cells. Pronounced anti-migratory activity, DNA fragmentation, decrease in expression of procaspase-3 and enhancement of p53 expression were further identified upon exposure to surface-coated IONs with tethered doxorubicin and ellipticine. Moreover, surface-coated IONs nanoformulations of topo II poisons exhibited exceptional stability in human plasma with no protein corona and complement 3 binding, and only a mild induction of hemolysis in human red blood cells. CONCLUSION: The results imply a high potential of an efficient ultrasound-mediated surface functionalization of IONs as delivery vehicles to improve therapeutic efficiency of topo II poisons.

• Keywords
• doxorubicin, ellipticine, iron oxide, nanoparticles, release kinetics,
• MeSH
• Coated Materials, Biocompatible chemistry   MeSH
• Cell Death drug effects   MeSH
• Cell Line MeSH
• DNA Topoisomerases, Type II metabolism   MeSH
• Doxorubicin pharmacology   MeSH
• Wound Healing drug effects   MeSH
• Topoisomerase II Inhibitors pharmacology   MeSH
• Kinetics MeSH
• Humans MeSH
• Nanoparticles chemistry   MeSH
• Cell Movement drug effects   MeSH
• Surface Properties MeSH
• Static Electricity MeSH
• Drug Liberation * MeSH
• Sonication methods   MeSH
• Ferric Compounds chemistry   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Coated Materials, Biocompatible MeSH
• DNA Topoisomerases, Type II MeSH
• Doxorubicin MeSH
• ferric oxide MeSH Browser
• Topoisomerase II Inhibitors MeSH
• Ferric Compounds MeSH

Doxorubicin (DOX) is one of the most frequently used anticancer drugs in breast cancer treatment. However, clinical applications of DOX are restricted, largely due to the fact that its action disturbs the pro/antioxidant balance in both cancerous and non-cancerous cells. The aim of this study was to investigate the influence of fullerene (C60) in cell treatment by DOX on the proliferation of human breast cancer cells (MCF-7), concentration of metallothionein (MT) and superoxide dismutase (SOD), and SOD activity in these cells. The use of C60 in complexes with DOX causes a change in the level of cell proliferation of about 5% more than when caused by DOX alone (from 60⁻65% to 70%). The use of C60 as a DOX nanotransporter reduced the MT level increase induced by DOX. C60 alone caused an increase of SOD1 concentration. On the other hand, it led to a decrease of SOD activity. C60 in complex with DOX caused a decrease of the DOX-induced SOD activity level. Exposure of MCF-7 cells to DOX-C60 complexes results in a decrease in viable cells and may become a new therapeutic approach to breast cancer. The effects of C60 in complexes with DOX on MCF-7 cells included a decreased enzymatic (SOD activity) and nonenzymatic (MT) antioxidant status, thus indicating their prooxidant role in MCF-7 cells.

• Keywords
• breast tumors, doxorubicin, drug delivery systems, fullerene, nanoparticles, metallothionein, superoxide dismutase,
• MeSH
• Doxorubicin pharmacology   MeSH
• Fullerenes chemistry   MeSH
• Humans MeSH
• Metallothionein metabolism   MeSH
• MCF-7 Cells MeSH
• Nanoparticles chemistry   MeSH
• Cell Proliferation drug effects   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Superoxide Dismutase-1 metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Doxorubicin MeSH
• Fullerenes MeSH
• Metallothionein MeSH
• Antineoplastic Agents MeSH
• SOD1 protein, human MeSH Browser
• Superoxide Dismutase-1 MeSH

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.

• Keywords
• DNA damage, acetylation of histones, apoptosis, ellipticine, neuroblastoma, valproate,
• MeSH
• Apoptosis MeSH
• Ellipticines toxicity   MeSH
• Histone Deacetylase Inhibitors toxicity   MeSH
• Valproic Acid toxicity   MeSH
• Humans MeSH
• Mutagens toxicity   MeSH
• Cell Line, Tumor MeSH
• Neuroblastoma metabolism   MeSH
• Neurons drug effects   metabolism   MeSH
• Drug Synergism MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ellipticines MeSH
• ellipticine MeSH Browser
• Histone Deacetylase Inhibitors MeSH
• Valproic Acid MeSH
• Mutagens 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.

• Keywords
• Coenzymes, DNA, Enzymes, High pressure liquid chromatography,
• Publication type
• Journal Article 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
• Cytochrome P-450 CYP1A1 deficiency   genetics   metabolism   MeSH
• Ellipticines pharmacology   MeSH
• Hepatocytes drug effects   metabolism   MeSH
• Rats MeSH
• Mice MeSH
• DNA Damage * MeSH
• Antineoplastic Agents pharmacology   MeSH
• Uncoupling Agents pharmacology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Cytochrome P-450 CYP1A1 MeSH
• Ellipticines MeSH
• ellipticine MeSH Browser
• Antineoplastic Agents MeSH
• Uncoupling Agents MeSH

The requirements for early diagnostics as well as effective treatment of cancer diseases have increased the pressure on development of efficient methods for targeted drug delivery as well as imaging of the treatment success. One of the most recent approaches covering the drug delivery aspects is benefitting from the unique properties of nanomaterials. Ellipticine and its derivatives are efficient anticancer compounds that function through multiple mechanisms. Formation of covalent DNA adducts after ellipticine enzymatic activation is one of the most important mechanisms of its pharmacological action. In this study, we investigated whether ellipticine might be released from its micellar (encapsulated) form to generate covalent adducts analogous to those formed by free ellipticine. The (32)P-postlabeling technique was used as a useful imaging method to detect and quantify covalent ellipticine-derived DNA adducts. We compared the efficiencies of free ellipticine and its micellar form (the poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO) block copolymer, P 119 nanoparticles) to form ellipticine-DNA adducts in rats in vivo. Here, we demonstrate for the first time that treatment of rats with ellipticine in micelles resulted in formation of ellipticine-derived DNA adducts in vivo and suggest that a gradual release of ellipticine from its micellar form might produce the enhanced permeation and retention effect of this ellipticine-micellar delivery system.

• MeSH
• DNA Adducts chemistry   metabolism   MeSH
• Ellipticines administration & dosage   chemistry   pharmacokinetics   MeSH
• Rats MeSH
• Metabolic Clearance Rate MeSH
• Micelles MeSH
• Organ Specificity MeSH
• Rats, Wistar MeSH
• Drug Compounding methods   MeSH
• Antineoplastic Agents administration & dosage   chemistry   pharmacokinetics   MeSH
• Tissue Distribution MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Ellipticines MeSH
• ellipticine MeSH Browser
• Micelles MeSH
• Antineoplastic Agents MeSH

Doxorubicin is a commonly used antineoplastic agent in the treatment of many types of cancer. Little is known about the interactions of doxorubicin with cardiac biomolecules. Serious cardiotoxicity including dilated cardiomyopathy often resulting in a fatal congestive heart failure may occur as a consequence of chemotherapy with doxorubicin. The purpose of this study was to determine the effect of exposure to doxorubicin on the changes in major amino acids in tissue of cardiac muscle (proline, taurine, glutamic acid, arginine, aspartic acid, leucine, glycine, valine, alanine, isoleucine, threonine, lysine and serine). An in vitro interaction study was performed as a comparison of amino acid profiles in heart tissue before and after application of doxorubicin. We found that doxorubicin directly influences myocardial amino acid representation even at low concentrations. In addition, we performed an interaction study that resulted in the determination of breaking points for each of analyzed amino acids. Lysine, arginine, β-alanine, valine and serine were determined as the most sensitive amino acids. Additionally we compared amino acid profiles of myocardium before and after exposure to doxorubicin. The amount of amino acids after interaction with doxorubicin was significantly reduced (p = 0.05). This fact points at an ability of doxorubicin to induce changes in quantitative composition of amino acids in myocardium. Moreover, this confirms that the interactions between doxorubicin and amino acids may act as another factor most likely responsible for adverse effects of doxorubicin on myocardium.

• MeSH
• Amino Acids drug effects   metabolism   MeSH
• Chromatography, Ion Exchange MeSH
• Doxorubicin administration & dosage   adverse effects   MeSH
• Chickens MeSH
• Humans MeSH
• Myocardium metabolism   MeSH
• Neoplasms drug therapy   pathology   MeSH
• Drug-Related Side Effects and Adverse Reactions MeSH
• Antineoplastic Agents MeSH
• Heart drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Doxorubicin MeSH
• Antineoplastic Agents MeSH