BACKGROUND: Kaurane-type diterpenoids, obtained from various natural sources, have shown many biological activities, including anti-inflammatory and antitumor effects. Caracasine, an ent-kaurane diterpenoid isolated from the flowers of Croton micans, was shown to induce apoptosis in leukaemia cell lines. OBJECTIVE: The present study aimed to ascertain the compound's mechanism of cell death induction using two leukaemia cell lines, Jurkat E6.1 (T cell) and HL-60 (promyeloblast cells). METHODS: Cell death in Jurkat and HL60 cells were evaluated by flow cytometry for apoptosis with annexin-V/PI, mitochondrial membrane potential disturbance, changes in cell cycle, CD95 expression, caspase activation, Nuclear Factor kappa B inhibition, and differentiation into a neutrophil-like cell (dHL60). RESULTS: Caracasine (10 μM) increased the G0/G1 phase in Jurkat and arrested the cell cycle in the S phase in HL60. Caracasine increased CD95 expression (p<0.01 in Jurkat and p<0.05 in HL60) and caspase-8 activation (p<0.001 in Jurkat and p<0.05 in HL60). Caspase-9 was activated in both cell lines (p<0.001) along with the decline in mitochondrial Δψm (p<0.05 in Jurkat and p<0.001 in HL60). In HL60 cells, the kaurane induced neutrophil differentiation was assessed by CD40 expression and reactive oxygen species production. In Jurkat cells, caracasine inhibited the NF-κB pathway in cells pretreated with PHA to activate the NF-κB pathway, suggesting a possible role in inflammatory diseases. CONCLUSION: Caracasine induced apoptosis through the intrinsic and extrinsic pathways in both cell lines were evaluated which could be the leading structure for new anti-leukemic and anti-inflammatory drugs.
- Klíčová slova
- Caracasine, NF-κB, apoptosis, caracasine acid, caspases, cell cycle, differentiation, kaurane-type diterpenoids,
- MeSH
- apoptóza MeSH
- diterpeny kauranové * farmakologie chemie MeSH
- diterpeny * farmakologie MeSH
- HL-60 buňky MeSH
- Jurkat buňky MeSH
- leukemie * farmakoterapie MeSH
- lidé MeSH
- NF-kappa B metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- diterpeny kauranové * MeSH
- diterpeny * MeSH
- NF-kappa B MeSH
N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.
- Klíčová slova
- HL-60 cells, MPO, N-acetylcysteine, NOX, SOD, U937 cells, oxidative stress,
- MeSH
- acetylcystein farmakologie MeSH
- HL-60 buňky MeSH
- katalasa genetika MeSH
- leukemie farmakoterapie genetika metabolismus MeSH
- lidé MeSH
- NADPH-oxidasa 2 genetika MeSH
- oxidační stres účinky léků MeSH
- peroxidasa genetika MeSH
- proliferace buněk účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- stanovení celkové genové exprese MeSH
- superoxiddismutasa genetika MeSH
- U937 buňky MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylcystein MeSH
- CYBB protein, human MeSH Prohlížeč
- katalasa MeSH
- MPO protein, human MeSH Prohlížeč
- NADPH-oxidasa 2 MeSH
- peroxidasa MeSH
- reaktivní formy kyslíku MeSH
- superoxiddismutasa MeSH
Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.
- Klíčová slova
- HL-60 cells, NADPH oxidase, NOX4, U-937 cells, macrophages, phorbol 12-myristate 13-acetate, protein-centered radicals,
- MeSH
- acetofenony farmakologie MeSH
- barvení a značení MeSH
- buněčná diferenciace * účinky léků MeSH
- elektronová paramagnetická rezonance MeSH
- HL-60 buňky MeSH
- hydroxylový radikál MeSH
- lidé MeSH
- monocyty cytologie účinky léků metabolismus MeSH
- NADP metabolismus MeSH
- proliferace buněk účinky léků MeSH
- proteiny metabolismus MeSH
- tetradekanoylforbolacetát farmakologie MeSH
- tvar buňky účinky léků MeSH
- U937 buňky MeSH
- viabilita buněk účinky léků MeSH
- volné radikály metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetofenony MeSH
- acetovanillone MeSH Prohlížeč
- hydroxylový radikál MeSH
- NADP MeSH
- proteiny MeSH
- tetradekanoylforbolacetát MeSH
- volné radikály MeSH
The anthracycline (ANT) anticancer drugs such as doxorubicin or daunorubicin (DAU) can cause serious myocardial injury and chronic cardiac dysfunction in cancer survivors. A bisdioxopiperazine agent dexrazoxane (DEX) has been developed as a cardioprotective drug to prevent these adverse events, but it is uncertain whether it is the best representative of the class. The present study used a rabbit model of chronic ANT cardiotoxicity to examine another bisdioxopiperazine compound called GK-667 (meso-(butane-2,3-diylbis(2,6-dioxopiperazine-4,1-diyl))bis(methylene)-bis(2-aminoacetate) hydrochloride), a water-soluble prodrug of ICRF-193 (meso-4,4'-(butan-2,3-diyl)bis(piperazine-2,6-dione)), as a potential cardioprotectant. The cardiotoxicity was induced by DAU (3 mg/kg, intravenously, weekly, 10 weeks), and GK-667 (1 or 5 mg/kg, intravenously) was administered before each DAU dose. The treatment with GK-667 was well tolerated and provided full protection against DAU-induced mortality and left ventricular (LV) dysfunction (determined by echocardiography and LV catheterization). Markers of cardiac damage/dysfunction revealed minor cardiac damage in the group co-treated with GK-667 in the lower dose, whereas almost full protection was achieved with the higher dose. This was associated with similar prevention of DAU-induced dysregulation of redox and calcium homeostasis proteins. GK-667 dose-dependently prevented tumor suppressor p53 (p53)-mediated DNA damage response in the LV myocardium not only in the chronic experiment but also after single DAU administration. These effects appear essential for cardioprotection, presumably because of the topoisomerase IIβ (TOP2B) inhibition provided by its active metabolite ICRF-193. In addition, GK-667 administration did not alter the plasma pharmacokinetics of DAU and its main metabolite daunorubicinol (DAUol) in rabbits in vivo. Hence, GK-667 merits further investigation as a promising drug candidate for cardioprotection against chronic ANT cardiotoxicity.
- Klíčová slova
- ICRF-193, anthracycline cardiotoxicity, bisdioxopiperazine, cardioprotection, dexrazoxane, topoisomerase IIbeta,
- MeSH
- chronická nemoc MeSH
- daunomycin MeSH
- diketopiperaziny farmakologie MeSH
- dysfunkce levé srdeční komory metabolismus patofyziologie prevence a kontrola MeSH
- fibróza MeSH
- funkce levé komory srdeční účinky léků MeSH
- HL-60 buňky MeSH
- inhibitory topoisomerasy II farmakologie MeSH
- kardiomyocyty účinky léků metabolismus patologie MeSH
- kardiomyopatie chemicky indukované metabolismus patofyziologie prevence a kontrola MeSH
- kardiotoxicita MeSH
- králíci MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- poškození DNA * MeSH
- prekurzory léčiv farmakologie MeSH
- remodelace komor účinky léků MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione MeSH Prohlížeč
- daunomycin MeSH
- diketopiperaziny MeSH
- inhibitory topoisomerasy II MeSH
- nádorový supresorový protein p53 MeSH
- prekurzory léčiv MeSH
Current multiagent chemotherapy regimens have improved the cure rate in acute leukemia patients, but they are highly toxic and poorly efficient in relapsed patients. To improve the treatment approaches, new specific molecules are needed. The G-quadruplexes (G4s), which are noncanonical nucleic acid structures found in specific guanine-rich DNA or RNA, are involved in many cellular events, including control of gene expression. G4s are considered as targets for the development of anticancer agents. Heterocyclic molecules are well known to target and stabilize G4 structures. Thus, a new series of 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives (1a-i) was designed, synthesized, and evaluated against five human myeloid leukemia cell lines (K562, KU812, MV4-11, HL60, and U937). Their ability to stabilize various oncogene promoter G4 structures (c-MYC, BCL-2, and K-RAS) as well as the telomeric G4 was also determined through the fluorescence resonance energy transfer melting assay and native mass spectrometry. In addition, the more bioactive ligands 1g-i were tested for telomerase activity in HuT78 and MV4-11 protein extracts.
- Klíčová slova
- 1, 10-phenanthroline, FRET melting, G-quadruplex, G4 ligands, antiproliferative activity, leukemia,
- MeSH
- akutní myeloidní leukemie farmakoterapie patologie MeSH
- antitumorózní látky chemická syntéza chemie farmakologie MeSH
- buňky K562 MeSH
- fenantroliny chemická syntéza chemie farmakologie MeSH
- G-kvadruplexy účinky léků MeSH
- HL-60 buňky MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- racionální návrh léčiv MeSH
- rezonanční přenos fluorescenční energie MeSH
- telomerasa metabolismus MeSH
- U937 buňky MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 1,10-phenanthroline MeSH Prohlížeč
- antitumorózní látky MeSH
- fenantroliny MeSH
- ligandy MeSH
- telomerasa MeSH
Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.
- Klíčová slova
- BCR-ABL1, CML, Quality, RTddPCR, external quality assessment (EQA),
- MeSH
- bcr-abl fúzové proteiny krev MeSH
- buňky K562 chemie MeSH
- chronická myeloidní leukemie krev MeSH
- HL-60 buňky chemie MeSH
- klinické laboratoře MeSH
- lidé MeSH
- lineární modely MeSH
- nádorové biomarkery krev MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- reagenční diagnostické soupravy MeSH
- reprodukovatelnost výsledků MeSH
- testování odbornosti laboratoří * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Asie MeSH
- Evropa MeSH
- Severní Amerika MeSH
- Názvy látek
- bcr-abl fúzové proteiny MeSH
- BCR-ABL1 fusion protein, human MeSH Prohlížeč
- nádorové biomarkery MeSH
- reagenční diagnostické soupravy MeSH
The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.
- Klíčová slova
- Bivariate methods, Cell cycle analysis, DNA labeling, Replication,
- MeSH
- barvení a značení metody MeSH
- bromodeoxyuridin aplikace a dávkování MeSH
- buněčný cyklus * MeSH
- DNA analýza MeSH
- fluorescenční barviva aplikace a dávkování MeSH
- HeLa buňky MeSH
- HL-60 buňky MeSH
- Jurkat buňky MeSH
- karbocyaniny aplikace a dávkování MeSH
- lidé MeSH
- průtoková cytometrie metody MeSH
- replikace DNA * MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bromodeoxyuridin MeSH
- cyanine dye 3 MeSH Prohlížeč
- DNA MeSH
- fluorescenční barviva MeSH
- karbocyaniny MeSH
Continuing our studies aimed at A-ring modified vitamin D compounds, we designed novel 19-norcalcitriol derivatives bearing at C-2 pegylated chains of different lengths. The terminal fragments of these substituents contain hydroxyls or moieties possessing nitrogen and/or sulfur atoms capable of transition metal ions complexation. Also, two conjugate-type platinum(II) complexes of 19-norcalcitriol were obtained in which l-methionine served as chelating moiety. The convergent synthesis of the target 19-norcalcitriol analogs involved several steps with the crucial one being condensation of A-ring phosphine oxide and the known Grundmann ketone by Wittig-Horner reaction. Further elaboration of the 2-alkylidene substituent provided all final compounds which were then tested to determine their affinity for the vitamin D receptor and cytotoxic activity.
- Klíčová slova
- 19-Norcalcitriol, Drug delivery systems, Platinum(II) complexes, Vitamin D receptor, Wittig-Horner reaction,
- MeSH
- antitumorózní látky farmakologie MeSH
- HL-60 buňky MeSH
- kalcitriol chemická syntéza chemie farmakologie MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- ligandy MeSH
- MFC-7 buňky MeSH
- preklinické hodnocení léčiv MeSH
- racionální návrh léčiv * MeSH
- receptory kalcitriolu účinky léků MeSH
- simulace molekulového dockingu MeSH
- vazebná místa MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antitumorózní látky MeSH
- kalcitriol MeSH
- ligandy MeSH
- receptory kalcitriolu MeSH
Bisdioxopiperazine agent dexrazoxane (ICRF-187) has been the only effective and approved drug for prevention of chronic anthracycline cardiotoxicity. However, the structure-activity relationships (SARs) of its cardioprotective effects remain obscure owing to limited investigation of its derivatives/analogs and uncertainties about its mechanism of action. To fill these knowledge gaps, we tested the hypothesis that dexrazoxane derivatives exert cardioprotection via metal chelation and/or modulation of topoisomerase IIβ (Top2B) activity in chronic anthracycline cardiotoxicity. Dexrazoxane was alkylated in positions that should not interfere with the metal-chelating mechanism of cardioprotective action; that is, on dioxopiperazine imides or directly on the dioxopiperazine ring. The protective effects of these agents were assessed in vitro in neonatal cardiomyocytes. All studied modifications of dexrazoxane molecule, including simple methylation, were found to abolish the cardioprotective effects. Because this challenged the prevailing mechanistic concept and previously reported data, the two closest derivatives [(±)-4,4'-(propane-1,2-diyl)bis(1-methylpiperazine-2,6-dione) and 4-(2-(3,5-dioxopiperazin-1-yl)ethyl)-3-methylpiperazine-2,6-dione] were thoroughly scrutinized in vivo using a rabbit model of chronic anthracycline cardiotoxicity. In contrast to dexrazoxane, both compounds failed to protect the heart, as demonstrated by mortality, cardiac dysfunction, and myocardial damage parameters, although the pharmacokinetics and metal-chelating properties of their metabolites were comparable to those of dexrazoxane. The loss of cardiac protection was shown to correlate with their abated potential to inhibit and deplete Top2B both in vitro and in vivo. These findings suggest a very tight SAR between bisdioxopiperazine derivatives and their cardioprotective effects and support Top2B as a pivotal upstream druggable target for effective cardioprotection against anthracycline cardiotoxicity. SIGNIFICANCE STATEMENT: This study has revealed the previously unexpected tight structure-activity relationships of cardioprotective effects in derivatives of dexrazoxane, which is the only drug approved for the prevention of cardiomyopathy and heart failure induced by anthracycline anticancer drugs. The data presented in this study also strongly argue against the importance of metal-chelating mechanisms for the induction of this effect and support the viability of topoisomerase IIβ as an upstream druggable target for effective and clinically translatable cardioprotection.
- MeSH
- antracykliny škodlivé účinky MeSH
- dexrazoxan farmakologie MeSH
- DNA-topoisomerasy typu II metabolismus MeSH
- HL-60 buňky MeSH
- inhibitory topoisomerasy II farmakologie MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- kardiomyopatie farmakoterapie metabolismus MeSH
- kardiotoxicita farmakoterapie MeSH
- králíci MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- modely u zvířat MeSH
- myokard metabolismus MeSH
- nádorové buněčné linie MeSH
- ochranné látky farmakologie MeSH
- potkani Wistar MeSH
- srdce účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antracykliny MeSH
- dexrazoxan MeSH
- DNA-topoisomerasy typu II MeSH
- inhibitory topoisomerasy II MeSH
- ochranné látky MeSH
A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea 12-17 and urea heterodimers 18-22, and to measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mitochondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14-17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells.
- Klíčová slova
- 7-MEOTA-tacrine heterodimers, HL-60, calf thymus DNA, human dermal fibroblasts, topoisomerases,
- MeSH
- akridiny chemická syntéza chemie farmakologie MeSH
- antitumorózní látky chemická syntéza chemie farmakologie MeSH
- buňky A549 MeSH
- fibroblasty účinky léků MeSH
- HL-60 buňky MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- proliferace buněk účinky léků MeSH
- takrin chemie farmakologie MeSH
- thiomočovina chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 7-methoxy-1,2,3,4-tetrahydroacridin-9-amine MeSH Prohlížeč
- akridiny MeSH
- antitumorózní látky MeSH
- takrin MeSH
- thiomočovina MeSH