Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 μM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.

• MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza * účinky léků   MeSH
• bcr-abl fúzové proteiny * genetika   antagonisté a inhibitory   metabolismus   MeSH
• chemorezistence * účinky léků   MeSH
• chronická myeloidní leukemie * farmakoterapie   genetika   patologie   MeSH
• dasatinib farmakologie   MeSH
• imatinib mesylát * farmakologie   terapeutické užití   MeSH
• inhibitory proteinkinas * farmakologie   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• oligonukleotidy * farmakologie   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL oncogene. Despite the high performance of treatment with tyrosine kinase inhibitors (TKI), about 30% of patients develop resistance to the therapy. To improve the outcomes, identification of new targets of treatment is needed. Here, we explored the Casein Kinase 2 (CK2) as a potential target for CML therapy. Previously, we detected increased phosphorylation of HSP90β Serine 226 in patients non-responding to TKIs imatinib and dasatinib. This site is known to be phosphorylated by CK2, which was also linked to CML resistance to imatinib. In the present work, we established six novel imatinib- and dasatinib-resistant CML cell lines, all of which had increased CK2 activation. A CK2 inhibitor, CX-4945, induced cell death of CML cells in both parental and resistant cell lines. In some cases, CK2 inhibition also potentiated the effects of TKI on the cell metabolic activity. No effects of CK2 inhibition were observed in normal mononuclear blood cells from healthy donors and BCR-ABL negative HL60 cell line. Our data indicate that CK2 kinase supports CML cell viability even in cells with different mechanisms of resistance to TKI, and thus represents a potential target for treatment.

• MeSH
• apoptóza MeSH
• bcr-abl fúzové proteiny metabolismus   MeSH
• buněčná smrt MeSH
• chemorezistence MeSH
• chronická myeloidní leukemie * farmakoterapie   MeSH
• dasatinib farmakologie   MeSH
• imatinib mesylát farmakologie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• inhibitory tyrosinkinasy MeSH
• kaseinkinasa II * MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Long-chain n-3 polyunsaturated fatty acids modulate immune cell functions. The primary objective of this study was to evaluate the impact of different lipid emulsions (LEs) with supplemented doses of fish oil (FO) on serum cytokine concentration and in vitro cytokine production in patients with intestinal failure on home parenteral nutrition (HPNPs). We hypothesized that FO supplementation would diminish lipopolysaccharide (LPS)-stimulated cytokine production. Twelve HPNPs receiving Smoflipid for at least 3 months were given FO (Omegaven) for a further 4 weeks. After this cycle, the patients were randomized to subsequently receive 1 cycle with Lipoplus and 1 cycle with ClinOleic for 6 weeks or vice versa plus 4 weeks of added Omegaven after each cycle in a crossover design. Comparison of the baseline LE regimens showed lower LPS-stimulated production of IL-1β in the HPNPs on Lipoplus than on the Smoflipid and ClinOleic regimens, as well as lower IL-8 compared to the Smoflipid regimen. Omegaven reduced IL-8 concentration in serum under the Lipoplus regimen and diminished LPS-stimulated production of IL-1β under the Smoflipid and ClinOleic. IL-6 and TNF-α production was depressed only in those on Smoflipid. Irrespective of the LE used, the HPNPs compared to the healthy controls showed higher IL-6, IL-8, and TNF-α concentrations in serum and LPS-stimulated production of IL-6 as well as lower n-6/n-3 long-chain polyunsaturated fatty acids in the erythrocyte phospholipids. LPS-stimulated production of IL-6 correlated negatively with the parenteral dose of eicosapentaenoic acid + docosahexaenoic acid. In conclusion, FO-supplemented parenteral nutrition suppresses in vitro cytokine production.

• MeSH
• cytokiny krev   MeSH
• dospělí MeSH
• imunologické faktory MeSH
• imunomodulace MeSH
• klinické křížové studie MeSH
• lidé MeSH
• lipopolysacharidy imunologie   MeSH
• parenterální výživa doma * metody   MeSH
• potravní doplňky MeSH
• rybí oleje * imunologie   MeSH
• techniky in vitro MeSH
• tukové emulze intravenózní MeSH
• zánět MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• Publikační typ
• klinická studie MeSH
• práce podpořená grantem MeSH

Long-chain n-3 polyunsaturated fatty acids modulate immune cell functions. The primary objective of this study was to evaluate the impact of different lipid emulsions (LEs) with supplemented doses of fish oil (FO) on serum cytokine concentration and in vitro cytokine production in patients with intestinal failure on home parenteral nutrition (HPNPs). We hypothesized that FO supplementation would diminish lipopolysaccharide (LPS)-stimulated cytokine production. Twelve HPNPs receiving Smoflipid for at least 3 months were given FO (Omegaven) for a further 4 weeks. After this cycle, the patients were randomized to subsequently receive 1 cycle with Lipoplus and 1 cycle with ClinOleic for 6 weeks or vice versa plus 4 weeks of added Omegaven after each cycle in a crossover design. Comparison of the baseline LE regimens showed lower LPS-stimulated production of IL-1β in the HPNPs on Lipoplus than on the Smoflipid and ClinOleic regimens, as well as lower IL-8 compared to the Smoflipid regimen. Omegaven reduced IL-8 concentration in serum under the Lipoplus regimen and diminished LPS-stimulated production of IL-1β under the Smoflipid and ClinOleic. IL-6 and TNF-α production was depressed only in those on Smoflipid. Irrespective of the LE used, the HPNPs compared to the healthy controls showed higher IL-6, IL-8, and TNF-α concentrations in serum and LPS-stimulated production of IL-6 as well as lower n-6/n-3 long-chain polyunsaturated fatty acids in the erythrocyte phospholipids. LPS-stimulated production of IL-6 correlated negatively with the parenteral dose of eicosapentaenoic acid + docosahexaenoic acid. In conclusion, FO-supplemented parenteral nutrition suppresses in vitro cytokine production.

• MeSH
• cytokiny krev   MeSH
• dospělí MeSH
• klinické křížové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• parenterální výživa doma metody   MeSH
• potravní doplňky MeSH
• rybí oleje aplikace a dávkování   krev   farmakologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• techniky in vitro MeSH
• tukové emulze intravenózní aplikace a dávkování   metabolismus   farmakologie   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH

OBJECTIVE: Murine erythroleukemia (MEL) cells are transformed erythroid precursors that are arrested in an immature and proliferating state. These leukemic cells can be grown in cell cultures and induced to terminal erythroid differentiation by a treatment with a specific chemical inducer such as N,N'-hexamethylene bisacetamide. MEL cells then re-enter their original erythroid program and differentiate along the erythroid pathway into non-dividing hemoglobin-rich cells resembling orthochromatophilic normoblasts. To deepen our understanding of the molecular mechanisms underlying and erythroid differentiation and leukemia we monitored changes in protein expression in differentiating MEL cells. METHODS: In our effort to find new candidate proteins involved in the differentiation of MEL cells, we embraced a proteomic approach. Employing two-dimensional (2D) electrophoresis combined with mass spectrometry, we compared protein expression in non-induced MEL cells with MEL cells exposed to N,N'-hexamethylene bisacetamide for 48 h. RESULTS: From 700 proteins spots observed, 31 proteins were differentially expressed. We successfully identified 27 of the differentially expressed molecules by mass spectrometry (MALDI-MS). CONCLUSION: In addition to proteins involved in heme biosynthesis, protein metabolism, stress defense and cytoskeletal organization, we identified 3 proteins engaged in regulation of cellular trafficking and 7 proteins important for regulation of gene expression and cell cycle progression including 3 components of chromatin remodeling complexes. Many of the identified molecules are associated with erythroid differentiation or leukemia for the first time. To our knowledge, this is the first study applying a modern proteomic approach to the direct analysis of erythroid differentiation of leukemic cells.

• MeSH
• 2D gelová elektroforéza metody   MeSH
• acetamidy farmakologie   MeSH
• akutní erytroblastická leukemie farmakoterapie   metabolismus   MeSH
• buněčná diferenciace účinky léků   MeSH
• erytroidní buňky chemie   metabolismus   účinky léků   MeSH
• financování organizované MeSH
• hmotnostní spektrometrie metody   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové buňky kultivované MeSH
• proteom chemie   metabolismus   účinky léků   MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

Hereditary hemochromatosis type I is an autosomal-recessive iron overload disease associated with a mutation in HFE gene. The most common mutation, C282Y, disrupts the disulfide bond necessary for the association of HFE with beta-2-microglobulin and abrogates cell surface HFE expression. HFE-deficient mice develop iron overload indicating a central role of the protein in the pathogenesis of hereditary hemochromatosis type I. However, despite significant effort, the role of the HFE protein in iron metabolism is still unknown. To shed a light on the molecular mechanism of HFE-related hemochromatosis we studied protein expression changes elicited by HFE-deficiency in the liver which is the organ critical for the regulation of iron metabolism. We undertook a proteomic study comparing protein expression in the liver of HFE deficient mice with control animals. We compared HFE-deficient animals with control animals with identical iron levels obtained by dietary treatment to identify changes specific to HFE deficiency rather than iron loading. We found 11 proteins that were differentially expressed in the HFE-deficient liver using two-dimensional electrophoresis and mass spectrometry identification. Of particular interest were urinary proteins 1, 2 and 6, glutathione-S-transferase P1, selenium binding protein 2, sarcosine dehydrogenase and thioredoxin-like protein 2. Our data suggest possible involvement of lipocalins, TNF-alpha signaling and PPAR alpha regulatory pathway in the pathogenesis of hereditary hemochromatosis and suggest future targeted research addressing the roles of the identified candidate genes in the molecular mechanism of hereditary hemochromatosis.

• MeSH
• 2D gelová elektroforéza MeSH
• exprese genu MeSH
• financování organizované MeSH
• hemochromatóza genetika   metabolismus   patologie   MeSH
• histokompatibilita - antigeny třídy I fyziologie   genetika   MeSH
• játra metabolismus   MeSH
• membránové proteiny fyziologie   genetika   nedostatek   MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• polymerázová řetězová reakce MeSH
• přetížení železem genetika   metabolismus   patologie   MeSH
• proteom genetika   metabolismus   MeSH
• proteomika metody   MeSH
• sekvence aminokyselin MeSH
• spektrofotometrie atomová MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

Liver iron overload can be found in hereditary hemochromatosis, chronic liver diseases such as alcoholic liver disease, and chronic viral hepatitis or secondary to repeated blood transfusions. The excess iron promotes liver damage, including fibrosis, cirrhosis, and hepatocellular carcinoma. Despite significant research effort, we remain largely ignorant of the cellular consequences of liver iron overload and the cellular processes that result in the observed pathological changes. In addition, the variability in outcome and the compensatory response that likely modulates the effect of increased iron levels are not understood. To provide insight into these critical questions, we undertook a study to determine the consequences of iron overload on protein levels in liver using a proteomic approach. Using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we studied hepatic iron overload induced by carbonyl iron-rich diet in mice and identified 30 liver proteins whose quantity changes in condition of excess liver iron. Among the identified proteins were enzymes involved in several important metabolic pathways, namely the urea cycle, fatty acid oxidation, and the methylation cycle. This pattern of changes likely reflects compensatory and pathological changes associated with liver iron overload and provides a window into these processes.

• MeSH
• 2D gelová elektroforéza MeSH
• enzymy metabolismus   MeSH
• financování organizované MeSH
• játra enzymologie   fyziologie   metabolismus   MeSH
• mastné kyseliny metabolismus   MeSH
• metylace MeSH
• močovina metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nemoci jater enzymologie   etiologie   metabolismus   MeSH
• oxidace-redukce MeSH
• přetížení železem chemicky indukované   komplikace   metabolismus   MeSH
• proteomika metody   MeSH
• sloučeniny železa MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• stupeň závažnosti nemoci MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.

• MeSH
• 2D gelová elektroforéza MeSH
• aldehyddehydrogenasa genetika   MeSH
• financování organizované MeSH
• hepatocelulární karcinom metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• játra metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• nádorové buněčné linie MeSH
• oxidační stres MeSH
• peroxidace lipidů MeSH
• přetížení železem metabolismus   MeSH
• proteomika metody   MeSH
• sekvence aminokyselin MeSH
• shluková analýza MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH