Multidrug resistance (MDR) is often associated with overexpression of the P-glycoprotein (P-gp, ABCB1). It was demonstrated that the P-gp mediated efflux decreases the drug concentration in cancer cells which results in the failure of chemotherapy. However, the MDR phenotype in cancer cells obviously involves various mechanisms. Therefore, if we want to estimate a contribution of the P-gp expression to the MDR phenotype, a clear quantitative relationship between the intracellular drug level and cell sensitivity must be established. To achieve this goal, a sensitive and non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite N-desmethyl imatinib (CGP 74588) has been developed. The assay is based on an optimised extraction of cells with 4% formic acid after their separation from the growth medium by centrifugation through a layer of silicone oil. Cell extracts are subsequently analyzed by LC/MS/MS. Calibration curves were linear from 1 to 500 nmol/l for imatinib and from 2 to 500 nmol/l for CGP 74588, with correlation coefficients (r(2)) better than 0.998 and 0.996, respectively. The limit of quantitation (LOQ) was 1 nmol/l for imatinib and 2 nmol/l for CGP 74588. Our method has been successfully applied to the determination of intracellular levels of imatinib in sensitive K562 and their resistant variant, K562/Dox cells.

• MeSH
• biotest metody   MeSH
• buňky K562 MeSH
• chronická myeloidní leukemie metabolismus   MeSH
• lidé MeSH
• limita detekce MeSH
• piperaziny chemie   metabolismus   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• protinádorové látky chemie   metabolismus   MeSH
• pyrimidiny chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

Embryonic stem cells (ESCs) proliferate rapidly and have a unique cell-cycle structure with a very short G1 phase. Previous reports suggested that the rapid G1 phase progression of ESCs might be underpinned by high and precocious Cdk2 activity and that Cdk2 activity might be crucial for both cell-cycle regulation and cell-fate decisions in human ESCs. However, the actual role of Cdk2 in cell-cycle progression of mouse ESCs (mESCs) has not been elucidated. In this study, we investigated the effects of down-regulation of Cdk2 activity by olomoucine II in 2 mESC lines. Olomoucine II treatment significantly increased the G1 phase cell numbers, decreased the S phase cell numbers, and inhibited DNA replication in mESCs. In nocodazole-synchronized mESCs, we show that specific down-regulation of Cdk2 activity prolongs G1 phase progression. In addition, down-regulation of Cdk2 activity in mESCs established a somatic cell-like cell cycle and induced expression of differentiation markers. Our results suggest that high Cdk2 activity is essential for rapid G1 phase progression and establishment of ESC-specific cell-cycle structure in mESCs and support the hypothesis of a link between cell-cycle regulation and pluripotency maintenance in ESCs. This study reveals olomoucine II to be an effective tool for manipulation of the cell cycle and pluripotency in ESCs and very likely also for the manipulation of other stem cell types, including cancer stem cells.

• MeSH
• buněčné linie MeSH
• buněčný cyklus účinky léků   MeSH
• buňky HT-29 MeSH
• časové faktory MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   genetika   metabolismus   MeSH
• embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• G1 fáze účinky léků   MeSH
• inbrední kmeny myší MeSH
• inhibiční koncentrace 50 MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteinkinasa CDC2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• průtoková cytometrie MeSH
• puriny farmakologie   MeSH
• replikace DNA účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakty MeSH

• Publikační typ
• abstrakty MeSH

AIM: The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) was studied. METHODS: To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed. RESULTS: We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resistance phenotype because of overexpression of P-gp, were significantly more resistant. We found that the intracellular level of IB-MECA in K562/HHT cells was approx. 10 times lower than those in HL-60 or K562 cells. Inhibitors of P-gp, including cyclosporine A (CsA) and verapamil (Vpa), increased the intracellular level of IB-MECA and reversed the resistance of K562/HHT cells to this drug. Accordingly, shRNA-mediated down-regulation of P-gp significantly increased the intracellular level of IB-MECA in K562/HHT cells which simultaneously exhibited reduced resistance to this A3AR agonist. In addition, an in vitro enzyme-based assay provided evidence that IB-MECA might serve as a substrate for P-gp. CONCLUSION: Our results suggest that P-gp overexpression prevents cells from IB-MECA induced apoptosis despite the A3AR expression. Pro-apoptotic effect of IB-MECA seemed to strongly depend on its intracellular accumulation rather than on its interaction with A3AR.

• MeSH
• adenosin analogy a deriváty   chemie   metabolismus   MeSH
• adenosintrifosfatasy metabolismus   MeSH
• agonisté adenosinového receptoru A3 MeSH
• apoptóza fyziologie   MeSH
• blokátory kalciových kanálů farmakologie   MeSH
• buňky K562 účinky léků   metabolismus   MeSH
• HL-60 buňky účinky léků   metabolismus   MeSH
• lidé MeSH
• mnohočetná léková rezistence fyziologie   MeSH
• P-glykoprotein antagonisté a inhibitory   genetika   metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• verapamil farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

The benzo[c]phenanthridine alkaloid sanguinarine has been studied for its antiproliferative activity in many cell types. Almost nothing however, is known about the cytotoxic effects of dihydrosanguinarine, a metabolite of sanguinarine. We compared the cytotoxicity of sanguinarine and dihydrosanguinarine in human leukemia HL-60 cells. Sanguinarine produced a dose-dependent decline in cell viability with IC(50) (inhibitor concentration required for 50% inhibition of cell viability) of 0.9 microM as determined by MTT assay after 4h exposure. Dihydrosanguinarine showed much less cytotoxicity than sanguinarine: at the highest concentration tested (20 microM) and 24h exposure, dihydrosanguinarine decreased viability only to 52%. Cytotoxic effects of both alkaloids were accompanied by activation of the intrinsic apoptotic pathway since we observed the dissipation of mitochondrial membrane potential, induction of caspase-9 and -3 activities, the appearance of sub-G(1) DNA and loss of plasma membrane asymmetry. This aside, sanguinarine also increased the activity of caspase-8. As shown by flow cytometry using annexin V/propidium iodide staining, 0.5 microM sanguinarine induced apoptosis while 1-4 microM sanguinarine caused necrotic cell death. In contrast, dihydrosanguinarine at concentrations from 5 microM induced primarily necrosis, whereas apoptosis occurred at 10 microM and above. We conclude that both alkaloids may cause, depending on the alkaloid concentration, both necrosis and apoptosis of HL-60 cells.

• MeSH
• apoptóza účinky léků   MeSH
• benzofenantridiny farmakologie   MeSH
• buněčný cyklus účinky léků   MeSH
• HL-60 buňky MeSH
• isochinoliny farmakologie   MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

The effect of cyclosporin A (CsA) on imatinib treated Bcr-Abl positive K562 cells was studied. Similarly to other authors we found that imatinib induced apoptosis and erythroid differentiation in K562 cells. While its low concentrations induced predominantly erythroid differentiation, higher concentrations induced apoptosis. We found that CsA significantly potentiated cytotoxic effects of imatinib. A detailed analysis revealed that CsA shifted the balance between differentiation and apoptosis in favour of apoptosis. Our findings indicated that the observed effect of CsA was mediated neither through inhibition of ERK1/2 (extracellular signal-regulated kinases 1/2), nor through inhibition of p38 MAPK. We further observed that CsA might sensitise cells to apoptosis due to a changed cellular redox status as combined treatment of cells with imatinib and CsA resulted in a dramatic decrease of the ratio between reduced (GSH) and oxidised (GSSG) glutathione GSH/GSSG and in a significant suppression of thioredoxin reductase enzymatic activity. Our results indicated that K562 cells did not express detectable level of P-glycoprotein (P-gp). In addition, CsA did not affect significantly the intracellular level of imatinib. Therefore we excluded the possibility that CsA increased sensitivity of cells to imatinib by the inhibition of P-gp-mediated drug efflux or by another mechanism involving modulation of intracellular drug concentration.

• MeSH
• apoptóza účinky léků   MeSH
• bcr-abl fúzní proteiny analýza   MeSH
• buňky K562 MeSH
• cyklosporin farmakologie   MeSH
• extracelulárním signálem regulované MAP kinasy antagonisté a inhibitory   MeSH
• glutathion analýza   MeSH
• glutathiondisulfid analýza   MeSH
• imidazoly farmakologie   MeSH
• lidé MeSH
• P-glykoprotein analýza   fyziologie   MeSH
• piperaziny farmakologie   MeSH
• protinádorové látky farmakologie   MeSH
• pyridiny farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.

• MeSH
• apoptóza účinky léků   MeSH
• chloromethylketony aminokyselin farmakologie   MeSH
• financování organizované MeSH
• inhibitory enzymů farmakologie   MeSH
• inhibitory kaspas MeSH
• kaspasa 3 metabolismus   MeSH
• lidé MeSH
• proliferace buněk účinky léků   MeSH
• spotřeba kyslíku účinky léků   MeSH
• U937 buňky enzymologie   patologie   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH