BACKGROUND: Dysregulation of the balance between cell growth and death in the colonic epithelium is associated with cancer promotion. Understanding how cell death in this self-renewing tissue is regulated and how it is influenced by interaction of specific dietary components, especially fat and fibre, could lead to improved treatment and prevention strategies for cancer. AIM OF THE STUDY: The effects of two types of polyunsaturated fatty acids (PUFAs)--arachidonic (AA, 20:4, n-6) or docosahexaenoic (DHA, 22:6, n-3)--on the response of human colon adenocarcinoma HT-29 cells to sodium butyrate (NaBt) were investigated. METHODS: The parameters reflecting cell proliferation and cell death were studied together with oxidative response, mitochondrial membrane potential (MMP) and changes of selected regulatory molecules associated with cell cycle (p27(Kip1) and p21(Cip1/WAF1)) and apoptosis (caspase-3, caspase-9, poly (ADP-ribose) polymerase--PARP, Bcl-2, Bax, Bak,Mcl-1). RESULTS: We demonstrated that pre-treatment with either AA or DHA attenuated cell cycle arrest caused by NaBt which is associated with modulation of p27(Kip1), but not p21(Cip1/WAF1) protein expression. On the other hand, PUFAs sensitised HT-29 cells to NaBt-induced apoptosis. An increased amount of floating cells and cells in the subG(0)/G(1) population was associated with increased reactive oxygen species production, lipid peroxidation, decrease of MMP, activation of caspase-3 and -9, PARP cleavage, and decrease in the expression of antiapoptotic Mcl-1 protein. The observed effects were modulated by the addition of a protein synthesis inhibitor, cycloheximide, and partially reversed by the antioxidant Trolox. CONCLUSIONS: PUFAs may have beneficial effects in the colon enhancing apoptosis induced by NaBt. Alteration of cell membrane lipid composition and potentiation of oxidative processes accompanied by changes in mitochondria followed by stimulation of apoptotic cascade components play a role in these effects.

• MeSH
• adenokarcinom * metabolismus   MeSH
• apoptóza účinky léků   MeSH
• buněčný cyklus účinky léků   MeSH
• buňky HT-29 MeSH
• butyráty * metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• kaspasa 3 MeSH
• kaspasa 9 MeSH
• kaspasy metabolismus   účinky léků   MeSH
• kyseliny arachidonové aplikace a dávkování   MeSH
• kyseliny dokosahexaenové aplikace a dávkování   MeSH
• lidé MeSH
• membránové potenciály účinky léků   MeSH
• nádory tračníku * metabolismus   MeSH
• nenasycené mastné kyseliny * metabolismus   MeSH
• peroxidace lipidů účinky léků   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   účinky léků   MeSH
• protoonkogenní proteiny metabolismus   účinky léků   MeSH
• průtoková cytometrie MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• srovnávací studie MeSH

OBJECTIVES: The resistance of tumor cells to cisplatin remains a major cause of treatment failure in cancer patients. In this study, the ability of Pt(IV) complex with adamantylamine-LA-12 and its reduced counterpart with lower oxidation state Pt(II)-LA-9 to overcome intrinsic cisplatin resistance was investigated. METHODS: The ovarian adenocarcinoma SK-OV-3 cells were exposed to cisplatin, LA-9, or LA-12 for 72 h and the effects of drug concentrations that caused 10% or 50% inhibition of cell proliferation were determined. After 24-72 h of sustained exposure viability, apoptosis and inhibition of proliferation were analyzed. DNA synthesis and cell cycle analysis were performed simultaneously in order to determine the modulation of cell cycle after platinum complexes treatment. RESULTS: Lung Resistance-related Protein (LRP/MVP) was detected in SK-OV-3 cells but not in the other two ovarian cancer lines with different sensitivity to cisplatin. LRP/MVP overexpression may be an important factor contributing to intrinsic cisplatin resistance. Interestingly, Pt(IV) complex-LA-12 had approximately 2.7-fold lower IC(50) concentration than LA-9 or cisplatin in SK-OV-3 cells. Moreover, LA-12 caused persistent accumulation of cells in S-phase of the cell cycle while LA-9 and cisplatin treatment-induced S-phase arrest was transient and shifted to G(2)/M-phase at later intervals. Apoptosis seemed to be not the dominant type of cell death caused by such the derivatives, but it was the most intensive after LA-12 treatment. CONCLUSIONS: We found strong differences between effects of Pt(IV) complex-LA-12 and Pt(II) derivatives-LA-9 and cisplatin on cytokinetic parameters. Overall, LA-12 but not its reduced Pt(II) counterpart LA-9 is the compound effective in p53 null human ovarian cancer cells and it is able to overcome intrinsic cisplatin resistance in these cells.

• MeSH
• adenokarcinom farmakoterapie   metabolismus   patologie   MeSH
• amantadin analogy a deriváty   aplikace a dávkování   MeSH
• buněčný cyklus účinky léků   MeSH
• buňky - růstové procesy účinky léků   MeSH
• chemorezistence MeSH
• cisplatina aplikace a dávkování   MeSH
• DNA nádorová biosyntéza   MeSH
• financování organizované MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny biosyntéza   MeSH
• nádory vaječníků farmakoterapie   metabolismus   patologie   MeSH
• organoplatinové sloučeniny aplikace a dávkování   farmakologie   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• protokoly protinádorové kombinované chemoterapie farmakologie   MeSH
• vault ribonucleoprotein particles antagonisté a inhibitory   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic β-cells contribute to the development of type 2 diabetes, however, the molecular mechanisms involved are unclear. AIMS: In this study we have tested the role of caspase-2 and suggested ER stress mediator JNK in saturated fatty acid-induced apoptosis of the human pancreatic β-cells NES2Y. RESULTS: We found that stearic acid at apoptosis-inducing concentration activated ER stress signaling pathways, i.e. IRE1α, PERK and ATF6 pathways, in NES2Y cells. During stearic acid-induced apoptosis, JNK inhibition did not decrease the rate of apoptosis nor the activation of caspase-8, -9, -7 and -2 and PARP cleavage. In addition, inhibition of JNK activity did not affect CHOP expression although it did decrease the induction of BiP expression after stearic acid treatment. Caspase-2 silencing had no effect on PARP as well as caspase-8, -9 and -7 cleavage and the induction of CHOP expression, however, it also decreased the induction of BiP expression. Surprisingly, caspase-2 silencing was accompanied by increased phosphorylation of c-Jun. CONCLUSIONS: We have demonstrated that caspase-2 as well as JNK are not key players in apoptosis induction by saturated fatty acids in human pancreatic β-cells NES2Y. However, they appear to be involved in the modulation of saturated fatty acid-induced ER stress signaling, probably by a mechanism independent of c-Jun phosphorylation.

• MeSH
• apoptóza účinky léků   MeSH
• beta-buňky cytologie   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• JNK mitogenem aktivované proteinkinasy antagonisté a inhibitory   metabolismus   MeSH
• kaspasa 2 chemie   genetika   metabolismus   MeSH
• kaspasa 7 metabolismus   MeSH
• kaspasa 8 metabolismus   MeSH
• kaspasa 9 metabolismus   MeSH
• kyseliny stearové farmakologie   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• poly(ADP-ribosa)polymerasy metabolismus   MeSH
• proteiny teplotního šoku metabolismus   MeSH
• RNA interference MeSH
• signální transdukce účinky léků   MeSH
• stres endoplazmatického retikula účinky léků   MeSH
• transkripční faktor ATF6 metabolismus   MeSH
• transkripční faktor CHOP metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.

• MeSH
• algoritmy MeSH
• beta-katenin chemie   metabolismus   MeSH
• buněčná diferenciace MeSH
• chromatin metabolismus   MeSH
• fenotyp MeSH
• genetická transkripce * MeSH
• homeostáza MeSH
• hyperplazie MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• myši MeSH
• organoidy metabolismus   MeSH
• proliferace buněk MeSH
• restrukturace chromatinu MeSH
• sekvence nukleotidů MeSH
• signální transdukce MeSH
• střevní sliznice cytologie   MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (-)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (-)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, Ki 0.92 µM for testosterone, IC₅₀ 1.46 µM, Ki 2.52 µM for midazolam; (-)-ketoconazole IC₅₀ 0.90 µM, Ki 0.17 µM for testosterone, IC₅₀ 1.04 µM, Ki 1.51 µM for midazolam).

• MeSH
• biokatalýza účinky léků   MeSH
• buňky Hep G2 MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• genetická transkripce účinky léků   MeSH
• hepatocyty účinky léků   enzymologie   MeSH
• jaterní mikrozomy účinky léků   metabolismus   MeSH
• ketokonazol chemie   farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• reportérové geny MeSH
• stereoizomerie MeSH
• steroidní receptory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The expression of the transcription factor encoded by the Wilms tumor gene 1 (WT1) is associated with a variety of human cancers. WT1 protein has been reported to serve as a target antigen for tumor-specific immune responses. We observed that the immunization of mice with peptide vaccines derived from WT1 in a mixture with the CpG adjuvant (ODN 1826) by tattoo administration was superior to subcutaneous delivery of the peptides in combination with CpG formulated with the mineral oil adjuvant or a DNA vaccine or a recombinant vaccinia virus vaccine expressing the truncated WT1 protein. Tattooing with the WT1122-140 and WT1126-134 peptide elicited the response of WT1-specific interferon-γ-producing T cells. Peptide vaccine administered with a tattoo device had an antitumor effect on the growth of the prostate tumor cell line TRAMP-C2, provided that the transforming growth factor-β produced by tumor cells was neutralized by anti-TGFβ monoclonal antibody. The treatment of the tumor-bearing mice with 5-azadeoxycytidine or poly IC did not work in synergy with the peptide vaccine.

• MeSH
• adjuvancia imunologická MeSH
• azacytidin analogy a deriváty   farmakologie   terapeutické užití   MeSH
• injekce intradermální MeSH
• monoklonální protilátky imunologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty terapie   MeSH
• poly I-C farmakologie   terapeutické užití   MeSH
• proteiny WT1 aplikace a dávkování   imunologie   MeSH
• protinádorové látky farmakologie   terapeutické užití   MeSH
• protinádorové vakcíny aplikace a dávkování   MeSH
• subjednotkové vakcíny aplikace a dávkování   MeSH
• tetování MeSH
• transformující růstový faktor beta antagonisté a inhibitory   imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• apoptóza genetika   MeSH
• buněčný cyklus fyziologie   účinky léků   MeSH
• Jurkat buňky cytologie   fyziologie   účinky léků   MeSH
• kaspasa 1 fyziologie   MeSH
• poly(A)-polymerasa fyziologie   účinky léků   MeSH
• průtoková cytometrie metody   MeSH
• staurosporin toxicita   MeSH

Inhibitors of cyclin-dependent kinases 9 have been developed as potential anticancer drugs for the treatment of multiple myeloma. We have previously prepared a library of arylazo-3,5-diaminopyrazole inhibitors of CDKs. Here, we describe a novel member, AAP1742 (CDK9 inhibition with IC(50) = 0.28 μm), that reduces the viability of multiple myeloma cell lines in low micromolar concentrations. Consistent with inhibition of CDK9, AAP1742 decreases the phosphorylation of RNA polymerase II and inhibits mRNA synthesis of anti-apoptotic proteins Mcl-1, Bcl-2, and XIAP, followed by apoptosis in the RPMI-8226 cell line in a dose- and a time-dependent manner. These results are consistent with the biochemical profile of AAP1742 and further suggest cellular inhibition of CDK9 as a possible target for anticancer drugs.

• MeSH
• aktivace enzymů účinky léků   MeSH
• apoptóza účinky léků   MeSH
• azosloučeniny chemie   farmakologie   MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   metabolismus   MeSH
• down regulace účinky léků   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mnohočetný myelom metabolismus   patologie   MeSH
• nádorové buněčné linie MeSH
• protein MCL-1 genetika   metabolismus   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• protoonkogenní proteiny c-bcl-2 genetika   metabolismus   MeSH
• pyrazoly chemie   farmakologie   MeSH
• RNA-polymerasa II metabolismus   MeSH
• X-vázaný inhibitor apoptózy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

• MeSH
• beta-katenin biosyntéza   MeSH
• buněčná diferenciace účinky léků   MeSH
• cytokiny biosyntéza   MeSH
• epitelové buňky účinky léků   MeSH
• interleukin-18 biosyntéza   MeSH
• kaspasa 1 biosyntéza   MeSH
• krysa rodu rattus MeSH
• Lacticaseibacillus rhamnosus * MeSH
• Lactobacillus casei * MeSH
• messenger RNA biosyntéza   MeSH
• mikrofilamentové proteiny biosyntéza   MeSH
• probiotika farmakologie   MeSH
• regulace genové exprese účinky léků   MeSH
• střevní sliznice cytologie   účinky léků   MeSH
• subcelulární frakce metabolismus   MeSH
• toll-like receptory biosyntéza   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen-thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.

• MeSH
• Cupriavidus necator genetika   metabolismus   MeSH
• hydroxybutyráty metabolismus   MeSH
• kryoprotektivní látky metabolismus   MeSH
• organely metabolismus   MeSH
• polyestery metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   MeSH
• zmrazování MeSH
• Publikační typ
• časopisecké články MeSH