[Glycine-N-methyltransferase and Malignant Diseases of the Prostate]. / Glycin-N-metyltransferáza a nádorová onemocnení prostaty.

BACKGROUND: Prostate cancer (PC) constitutes a heterogeneous group of diseases with high prevalence rates that are still increasing, particularly in western countries. Since 1980, prostate specific antigen (PSA) and other diagnostic approaches have been used for PC screening; however, some of these approaches are often deemed painful and cause invasive damage of tissue. Therefore, molecular approaches to PC diagnosis are attracting increasing attention, potentially providing patients with less stressful situations and providing better diagnoses and even prognostic information. Recent metabolomic and genomic studies have suggested that biomolecules can be used as diagnostic or prognostic markers or as targets for the development of novel therapeutic modalities. One of these molecules is glycine-N-methyltransferase (GNMT), an enzyme that plays a pivotal role in the biochemical conversion of glycine to sarcosine. The link between this molecule (encoded by homonymous gene - GNMT) and PC has been confirmed at several levels, and thus GNMT can be considered a promising target for the development of advanced diagnostic and/or prognostic approaches. AIM: The aim of this study was to analyse the physiological role of GNMT and to examine in greater detail its connection with PC at different levels, including gene structure, gene expression, and metabolism, in which GNMT plays an important role, not only in controlling the methylation status of cells, but also the metabolism of folic acid and methionine. Last but not least, we discuss the importance of cellular methylation processes and the link between their aberrations and PC development.Key words: glycine - folic acid - metabolism - methylation - sarcosineThis work was supported by GA CR 16-18917S, League against Cancer Prague (project 2022015) and Czech Ministry of Health - RVO, UH Motol 00064203.The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 9. 2. 2016Accepted: 20. 3. 2016.

[Modern Nanomedicine in Treatment of Lung Carcinomas]. / Moderní nanomedicína v lécbe karcinomu plic.

BACKGROUNDS: Despite the fast development of new effective cytostatics and targeted therapy, the treatment efficiency of lung cancer is still insufficient. The systemic administration of drugs results in a decrease in drug concentrations in tumor site, particularly due to specific extracellular environment in lungs. Nanotransporters could serve as a platform, protecting a drug against these undesired effects, which may enhance its therapeutic index and reduce side effects of a drug. Moreover, nanotechnologies possess the potential to improve the diagnostics of lung cancer, and thus increase a survival rate of oncologic patients. AIM: The presented study is aimed to demonstrate the possibilities provided by nanotechnologies in the field of treatment and diagnostic of lung cancers and discuss the obstacles, which complicate a translation into clinical practice.

[Modern imaging techniques for anthracycline cytostatics -  review of the literature]. / Moderní zobrazovací techniky pro antracyklinová cytostatika -  literární prehled.

Anthracycline cytostatics can be observed at the level of organelles, cells and whole organisms due to their fluorescent properties. Imaging techniques based on detection of fluorescence can be used not only for observation of drug interaction with tumor cells, but also for targeting therapy of tumors with nanoparticles containing anthracycline cytostatics. Doxorubicin and daunorubicin, enclosed in liposomes, as representatives of nanoparticles suitable for targeted therapy, are used in clinical practice. The main advantage of liposomal drugs is to reduce the side effects due to differences in pharmacokinetics and distribution of the drug in the body. Due to the fact that all biological mechanisms of action of anthracycline drugs are not still fully understood, modern imaging techniques offer tool for in vivo studies of these mechanisms.

Cytochrome P450- and peroxidase-mediated oxidation of anticancer alkaloid ellipticine dictates its anti-tumor efficiency.

An antineoplastic alkaloid ellipticine is a prodrug, whose pharmacological efficiency is dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation in target tissues. The aim of this review was to summarize our knowledge on the molecular mechanisms of ellipticine action in the cancer cells. The CYP-mediated ellipticine metabolites 9-hydroxy- and 7-hydroxyellipticine and the product of ellipticine oxidation by peroxidases, the ellipticine dimer, are the detoxication metabolites of this compound. In contrast, two carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, derived from two activation ellipticine metabolites, 13-hydroxyellipticine and 12-hydroxyellipticine, generate two major deoxyguanosine adducts in DNA found in the 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 glioblastoma U87MG cells in vitro and in rat breast carcinoma in vivo. Formation of these covalent DNA adducts by ellipticine is the predominant mechanism of its cytotoxicity and anti-tumor activity to these cancer cell lines. Ellipticine is also an inducer of CYP1A, 1B1, and 3A4 enzymes in the cancer cells and/or in vivo in rats exposed to this compound, thus modulating its own pharmacological efficiencies. The study forms the basis to further predict the susceptibility of human cancers to ellipticine and suggests that this alkaloid for treatment in combination with CYP and/or peroxidase gene transfer increasing the anticancer potential of this prodrug. It also suggests ellipticine reactive metabolites 13-hydroxyellipticine and 12-hydroxyellipticine to be good candidates for targeting to tumors absent from the CYP and peroxidase activation enzymes.

Neuroblastoma stem cells - mechanisms of chemoresistance and histone deacetylase inhibitors.

Cancer stem cells (CSCs) form a small proportion of tumor cells that have stem cell properties: self-renewal capacity, the ability to develop into different lineages and proliferative potential. The interest in CSCs emerged from their expected role in initiation, progression and recurrence of many tumors. They are generally resistant to conventional chemotherapy and radiotherapy. There are two hypotheses about their origin: The first assumes that CSCs may arise from normal stem cells, and the second supposes that differentiated cells acquire the properties of CSCs. Both hypotheses are not mutually exclusive, as it is possible that CSCs have a diverse origin in different tumors. CD133+ cells (CD133 is marker of CSC in some tumors) isolated from NBL, osteosarcoma and Ewing sarcoma cell lines are resistant to cisplatin, carboplatin, etoposide and doxorubicin than the CD133- ones. Being resistant to chemotherapy, there were many attempts to target CSCs epigenetically including the use of histone deacetylase inhibitors. The diverse influence of valproic acid (histone deacetylase inhibitor) on normal and cancer stem cells was proved in different experiments. We have found an increase percentage of CD133+ NBL cells after their incubation with VPA in a dose that does not induce apoptosis. Further researches on CSCs and clinical application for their detection are necessary: (i) to define the CSC function in carcinogenesis, cancer development and their role in metastasis; (ii) to find a specific marker for CSCs in different tumors; (iii) to explain the role of different pathways that determine their behavior and (iv) to explain mechanisms of chemoresistance of CSCs.

Clinical importance of matrix metalloproteinases.

This review gives a brief summary on clinical applications of MMPs and their determination. Primarily, the activity of MMPs in cancer formation, development and metastasis is discussed. Further, survey on methods including fluorimetric methods, zymographies, Western-blotting, immunocapture assay, enzyme-linked immunosorbent assay, immunocytochemistry and immunohistochemistry, phage display, multiple-enzyme/multiple-reagent system, activity profiling, chronopotentiometric stripping analysis and imaging methods for detection and determination of MMPs follows (Fig. 3, Ref. 100).

Biotransformation enzymes in development of renal injury and urothelial cancer caused by aristolochic acid.

Ingestion of aristolochic acid (AA) is associated with the development of AA-nephropathy and Balkan endemic nephropathy, which are characterized by chronic renal failure, tubulointerstitial fibrosis, and urothelial cancer. Understanding which enzymes are involved in AA activation and/or detoxification is important in assessing susceptibility to AA. Xiao et al. demonstrate that hepatic cytochrome P450s in mice detoxicate AA and thereby protect kidney from injury. The relative contribution of enzymes activating AA to induce urothelial cancer in humans remains to be resolved.

Oxidation pattern of the anticancer drug ellipticine by hepatic microsomes - similarity between human and rat systems.

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of DNA adducts mediated by cytochrome P450 (CYP). We investigated the ability of CYP enzymes in rat, rabbit and human hepatic microsomes to oxidize ellipticine and evaluated suitable animal models mimicking its oxidation in humans. Ellipticine is oxidized by microsomes of all species to 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only rat microsomes generated the pattern of ellipticine metabolites reproducing that formed by human microsomes. While rabbit microsomes favored the production of ellipticine N(2)-oxide, human and rat microsomes predominantly formed 13-hydroxyellipticine. The species difference in expression and catalytic activities of individual CYPs in livers are the cause of these metabolic differences. Formation of 7-hydroxy- and 9-hydroxyellipticine was attributable to CYP1A in microsomes of all species. However, production of 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, the metabolites generating DNA adducts, was attributable to the orthologous CYPs only in rats and humans. CYP3A predominantly generates these metabolites in rat and human microsomes, while CYP2C3 activity prevails in microsomes of rabbits. The results underline the suitability of rat species as a model to evaluate human susceptibility to ellipticine.

Continuous aerobic phenol degradation by defined mixed immobilized culture in packed bed reactors.

A defined mixed culture of Pseudomonas putida, Commamonas testosteroni and Candida tropicalis was immobilized by adsorption on polyurethane foam, cocoa-fibers, expanded slate and sintered glass. Packed bed reactors were used for long-term continuous phenol biodegradations. Loading experiments were done to study the impact of the following parameters: (1) hydraulic retention time, (2) dissolved oxygen concentration, and (3) elimination of the oxygen limitation. After the acclimation period (approximately 10 d), the loading test with the individual packings showed the following maximum degradation rates: sintered glass 34, polyurethane foam 12, expanded slate 11.5, and cocoa-fibers 7.7 kg m(-3) d(-1). All these values were reached at a removal efficiency >99 % and with oxygen in excess. Under these conditions, the pH of the diluted unbuffered medium in the reactor effluent was 3.2-4.0 and no incompletely oxidized metabolic intermediates were found. The free cell concentration in the effluent increased after the phenol overloading time period.

Activation of carcinogens by peroxidase. Horseradish peroxidase-mediated formation of benzenediazonium ion from a non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) and its binding to DNA.

Horseradish peroxidase in the presence of hydrogen peroxide (HRP/H2O2) oxidizes a carcinogenic non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) to the ultimate carcinogen, which binds to calf thymus DNA. The principal product of Sudan I oxidation by the HRP/H2O2 system is the benzenediazonium ion. Minor products are hydroxy derivatives of Sudan I, in which the aromatic rings are hydroxylated. The principal oxidative product (the benzenediazonium ion) is responsible for the carcinogenicity of Sudan I, because this ion, formed from this azo dye, binds to DNA.

Metabolism of carcinogenic N-nitroso-N-methylaniline by purified cytochromes P450 2B1 and P450 2B2.

N-Nitroso-N-methylaniline (NMA) is an esophageal carcinogen in the rat. The in vitro enzymatic metabolism of NMA was investigated using cytochromes P450 2B1 and P450 2B2, isolated from liver microsomes of rats pretreated with phenobarbital (PB), reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. Formaldehyde is produced by both cytochromes P450 (P450). NMA is a better substrate for P450 2B1 than for P450 2B2. The maximal velocity (Vmax) values are 3.3 and 1.6 nmol HCHO/min per nmol P450 for P450 2B1 and P450 2B2, respectively. Beside formation of formaldehyde, aniline and p-aminophenol (p-AP) are found to be metabolites formed from NMA by both P450 isoenzymes. P450 2B1 also affords phenol, while none was found with the P450 2B2 isoenzyme. Phenol formation presumably arose from direct alpha-C-hydroxylation of NMA via a benzenediazonium ion (BDI) intermediate. The results suggest strongly that P450 2B1 catalyzes both alpha-C-hydroxylation and denitrosation of NMA while P450 2B2 catalyzes only denitrosation. Therefore, the P450 2B1 isoenzyme participates in the activation of NMA to the ultimate carcinogenic BDI.

A new way to carcinogenicity of azo dyes: the benzenediazonium ion formed from a non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene(Sudan I) by microsomal enzymes binds to deoxyguanosine residues of DNA.

1-Phenylazo-2-hydroxynaphthalene (Sudan I) activated by pre-incubation with microsomal enzymes of rat livers covalently binds to DNA from calf thymus. Benzenediazonium ion formed from Sudan I by activation with microsomal enzymes is the principal active metabolite, which binds to DNA. Enzymatic hydrolysis of modified (14C-labelled) DNA, followed by separation of deoxynucleosides on a Sephadex G-10 column revealed that deoxyguanosine is the principal target for the binding of activated Sudan I. The high-performance liquid chromatographic (HPLC) analysis indicate that probably more than one radioactive adduct of activated Sudan I with deoxyguanosine is formed.

Prostaglandin H synthase-medicated oxidation and binding to DNA of a detoxication metabolite of carcinogenic Sudan I, 1-(phenylazo)-2,6-dihydroxynaphthalene.

The metabolite of the carcinogenic azo dye Sudan I, 1-(phenylazo)-2,6-dihydroxynaphthalene (6-OH-Sudan I), which is considered to be the detoxification product of this dye is metabolized by prostaglandin H synthase (PHS) in the presence of arachidonic acid or H2O2 in vitro. The apparent Michaelis constant value for 6-OH-Sudan I as a substrate is 98.9 microM. 1-(Phenylazo)-2,6-naphthoquinone is a principal product of the 6-OH-Sudan I oxidation. This oxidation is inhibited by radical scavengers nitrosobenzene, ascorbate, glutathione and NADH. This indicates that PHS metabolizes 6-OH-Sudan I through a one-electron oxidation mechanism, giving rise to free radicals. During the PHS-mediated reaction, 6-OH-Sudan I is activated to metabolites binding to protein and DNA. The 32P-postlabeling analysis of DNA modified by activated 6-OH-Sudan I provides evidence that covalent binding to DNA is the principal type of DNA modification. The PHS-mediated binding of 6-OH-Sudan I to DNA presumably proceeds through formation of 1-(phenylazo)-2,6-naphthoquinone. The results suggest strongly that the C-hydroxylated derivative of Sudan I (6-OH-Sudan I) should be evaluated as a proximate carcinogenic metabolite, which may participate in the initiation of Sudan I-carcinogenesis in the urinary bladder.

The first identification of the benzenediazonium ion formation from a non-aminoazo dye, 1-phenylazo-2-hydroxynaphthalene (Sudan I) by microsomes of rat livers.

1-Phenylazo-2-hydroxynaphthalene (Sudan I) is converted by microsomal enzymes of rat livers in vitro to 5 products. Hepatic microsomes from 5,6-benzoflavone-treated rats are more effective for the metabolism of Sudan I than those from phenobarbital- or Sudan I alone-treated rats. Major products formed by microsomes are identified as the ring-hydroxyderivatives of benzene and naphthalene rings. The formation of the benzenediazonium ion evolved by oxidative splitting of the azo group of Sudan I by microsomal enzymes is also proved. The oxidative splitting of Sudan I by microsomal enzymes may be considered as the possible mechanism of the Sudan I activation to the ultimate carcinogen (benzenediazonium ion).

Peroxidase oxidizes N-nitrosomethylaniline to ultimate carcinogens(s) binding to DNA and transfer RNA in vitro.

Carcinogenic N-nitrosomethylaniline is oxidized in vitro by horseradish peroxidase in the presence of H2O2 to ultimate carcinogens, which bind to DNA and transfer RNA (tRNA). tRNA is more accessible for modification by the activated carcinogen studied. The modification of nucleic acid by N-nitrosomethylaniline metabolite(s) formed by peroxidase is inhibited by some compounds of physiological importance (ascorbate, glutathine) and by radical trapping agents (nitrosobenzene, methyl viologen). 32P-postlabeling assay of DNA and tRNA modified by N-nitrosomethylaniline activated by peroxidase shows covalent adduct formation with nucleic acids. The role of peroxidases in the activation of N-nitrosamines leading to organ and/or cell specificity of these carcinogens is discussed.

Direct evidence for the formation of deoxyribonucleotide adducts from carcinogenic N-nitroso-N-methylaniline revealed by the 32P-postlabeling technique.

N-Nitroso-N-methylaniline (NMA) is an esophageal carcinogen in the rat. NMA forms a benzenediazonium ion (BDI) during microsomal cytochrome P-450 2B1 (CYP2B1) catalyzed metabolism. Using the nuclease P1-enhanced version of the 32P-postlabeling assay we investigated the formation of adducts by NMA with deoxyadenosine 3'-monophosphate (dAp) and deoxyguanosine 3'-monophosphate (dGp). 32P-postlabeling analysis of dAp and dGp, which were modified by NMA activated with microsomes of rats pretreated with phenobarbital (PB), and directly labeled resulted in each case in the appearance of one single adduct spot. Quantitative analysis of adducts revealed that the extent of dGp modification by activated NMA was more than 23 times greater than the extent of modification of dAp. The results suggest strongly that BDI, derived from NMA by CYP2B1 present in PB microsomes, participates in the formation of dAp and dGp adducts.

Detoxication products of the carcinogenic azodye Sudan I (solvent yellow 14) bind to nucleic acids after activation by peroxidase.

The C-hydroxyderivatives of the carcinogenic dye Sudan I, 1-phenylazo-2,6-dihydroxynaphthalene and 1-(4-hydroxyphenylazo)-2-hydroxynaphthalene, which are considered to be detoxication products of this dye bind to DNA or tRNA after oxidation into active metabolites by peroxidase and H2O2 in vitro. The 32P-postlabeling analysis of DNA modified by active metabolites of both Sudan I derivatives provides evidence that the covalent binding to DNA is the principal type of DNA modification. Since the urinary bladder is rich in peroxidases, the participation of these enzymes in activation of detoxicating products of Sudan I may be involved in the initiation of Sudan I-carcinogenesis in this organ.

The anticancer agent ellipticine on activation by cytochrome P450 forms covalent DNA adducts.

Ellipticine is a potent antitumor agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Using [3H]-labeled ellipticine, we observed substantial microsome (cytochrome P450)-dependent binding of ellipticine to DNA. In rat, rabbit, minipig, and human microsomes, in reconstituted systems with isolated cytochromes P450 and in Supersomes containing recombinantly expressed human cytochromes P450, we could show that ellipticine forms a covalent DNA adduct detected by [32P]-postlabeling. The most potent human enzyme is CYP3A4, followed by CYP1A1, CYP1A2, CYP1B1, and CYP2C9. Another minor adduct is formed independent of enzymatic activation. The [32P]-postlabeling analysis of DNA modified by activated ellipticine confirms the covalent binding to DNA as an important type of DNA modification. The DNA adduct formation we describe is a novel mechanism for the ellipticine action and might in part explain its tumor specificity.

Oxidation of xenobiotics by plant microsomes, a reconstituted cytochrome P450 system and peroxidase: a comparative study.

The microsomal fraction from tulip bulbs (Tulipa fosteriana, L.) contains cytochrome P450 (CYP3, EC 1.14.14.1) and peroxidase (EC 1.11.1.7.) enzymes catalyzing the NADPH--and hydrogen peroxide--dependent oxidation of the xenobiotic substrates, N-nitrosodimethylamine (NDMA), N-nitrosomethylaniline (NMA), aminopyrine and 1-phenylazo 2-hydroxynaphthalene (Sudan I), respectively. Oxidation of these model xenobiotics has also been assessed in a reconstituted electron-transport chain with a partially purified CYP fraction, phospholipid and isolated tulip NADPH:CYP reductase (EC 1.6.2.4.). Peroxidase isolated from tulip bulbs (isoenzyme C) oxidizes these xenobiotics, too. Values of kinetic parameters (Km, Vmax), requirements for cofactors (NADPH, hydrogen peroxide), the effect of inhibitors and identification of products formed from the xenobiotics by the microsomal fraction, partially purified CYP and peroxidase C were determined. These data were used to estimate the participation of the CYP preparation and peroxidase C in oxidation of two out of the four studied xenobiotics (NMA, Sudan I) in tulip microsomes. Using such detailed study, we found that the CYP-dependent enzyme system is responsible for the oxidation of these xenobiotics in the microsomal fraction of tulip bulbs. The results demonstrate the progress in resolving the role of plant CYP and peroxidase enzymes in oxidation of xenobiotics.

alpha-Naphthoflavone acts as activator and reversible or irreversible inhibitor of rabbit microsomal CYP3A6.

This report describes the effect of alpha-naphthoflavone (alpha-NF), a known substrate, inhibitor and activator of several cytochromes P450 (CYP), on rabbit CYP3A6. Hepatic microsomes of rabbit pretreated with rifampicine (RIF), enriched with CYP3A6, as well as purified CYP3A6 reconstituted with isolated NADPH:CYP reductase were used as enzymatic systems in this study. The data from difference spectroscopy experiments showed that alpha-NF does yield a type I binding spectrum. This compound is oxidized by microsomal CYP3A6 into two metabolites (5,6-epoxide and trans-7,8-dihydrodiol). While alpha-NF is a substrate of CYP3A6, it also acts as an enzyme modulator. Under the conditions used, stimulation of 17beta-estradiol 2-hydroxylation by alpha-NF was observed. In contrast, this compound reversibly inhibited N-demethylation of erythromycin and tamoxifen, competitively with respect to these substrates, having the K(i) values of 51.5 and 18.0 microM, respectively. Moreover, alpha-NF was found to be an effective inactivator of progesterone and testosterone 6beta-hydroxylation catalyzed by CYP3A6 in RIF-microsomes. In addition, time- and concentration-dependent inactivation of human CYP3A4-mediated 6beta-hydroxylation of testosterone by alpha-NF, was determined. The inactivation of CYP3A6 followed pseudo-first-order kinetics and was dependent on both NADPH and alpha-NF. The concentrations required for half-maximal inactivation (K(i)) were 80.1 and 108.5 microM and the times required for half of the enzyme to be inactivated were 10.0 and 11.9 min for 6beta-hydroxylation of progesterone and testosterone, respectively. The loss of the enzyme activity was not recovered following dialysis, while 90% of the ability to form a reduced CO complex remained. This indicates the binding of alpha-NF to a CYP apoprotein molecule rather than to a heme moiety. Protection from inactivation was seen in the presence of all tested CYP3A substrates. Progesterone and testosterone protected CYP3A6 against inactivation competitively with respect to inactivator, erythromycin non-competitively and 17beta-estradiol showed a mixed type of protection. Here, we described for the first time that alpha-NF is capable of irreversible inhibition of microsomal rabbit CYP3A6 and human CYP3A4. The obtained results strongly suggest that the CYP3A active center contains at least two and probably three distinct binding sites for substrates.