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.
- MeSH
- acetylace MeSH
- histony metabolismus MeSH
- hypoxie buňky fyziologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- neuroblastom metabolismus patologie MeSH
- protoonkogenní proteiny c-myc biosyntéza MeSH
- regulace genové exprese u nádorů fyziologie MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- biologické modely MeSH
- elipticiny aplikace a dávkování farmakologie MeSH
- inhibitory histondeacetylas aplikace a dávkování farmakologie MeSH
- jaterní mikrozomy účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- kyselina valproová aplikace a dávkování farmakologie MeSH
- kyseliny hydroxamové aplikace a dávkování farmakologie MeSH
- lidé MeSH
- nádorové buňky kultivované MeSH
- nádory mozku farmakoterapie genetika patologie MeSH
- neuroblastom farmakoterapie genetika patologie MeSH
- poškození DNA MeSH
- preklinické hodnocení léčiv MeSH
- protokoly protinádorové kombinované chemoterapie farmakologie terapeutické užití MeSH
- výsledek terapie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- adukty DNA metabolismus MeSH
- apoptóza účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- cyklooxygenasa 1 biosyntéza MeSH
- cyklooxygenasa 2 biosyntéza MeSH
- elipticiny farmakologie MeSH
- financování organizované MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- peroxidasa biosyntéza MeSH
- protinádorové látky farmakologie MeSH
- systém (enzymů) cytochromů P-450 biosyntéza MeSH
- viabilita buněk účinky léků MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
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.
- MeSH
- adukty DNA * MeSH
- cytotoxicita imunologická * MeSH
- elipticiny * farmakokinetika MeSH
- HL-60 buňky * MeSH
- inhibitory angiogeneze analýza MeSH
- lidé MeSH
- MFC-7 buňky * MeSH
- NADPH-cytochrom c-reduktasa * MeSH
- neuroblastom MeSH
- protinádorové látky MeSH
- rozpřahující látky * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
