BACKGROUND AND PURPOSE: Lymphatic transport of drugs after oral administration is an important mechanism for absorption of highly lipophilic compounds. Direct measurement in lymph duct cannulated animals is the gold standard method, but non-invasive cycloheximide chylomicron flow blocking method has gained popularity recently. However, concerns about its reliability have been raised. The aim of this work was to investigate the validity of cycloheximide chylomicron flow blocking method for the evaluation of lymphatic transport using model compounds with high to very high lipophilicity, that is, abiraterone and cinacalcet. EXPERIMENTAL APPROACH: Series of pharmacokinetic studies were conducted with abiraterone acetate and cinacalcet hydrochloride after enteral/intravenous administration to intact, lymph duct cannulated and/or cycloheximide pre-treated rats. KEY RESULTS: Mean total absolute oral bioavailability of abiraterone and cinacalcet was 7.0% and 28.7%, respectively. There was a large and significant overestimation of the lymphatic transport extent by the cycloheximide method. Mean relative lymphatic bioavailability of abiraterone and cinacalcet in cycloheximide method was 28-fold and 3-fold higher than in cannulation method, respectively. CONCLUSION AND IMPLICATIONS: Cycloheximide chylomicron flow blocking method did not provide reliable results on lymphatic absorption and substantially overestimated lymphatic transport for both molecules, that is, abiraterone and cinacalcet. This non-invasive method should not be used for the assessment of lymphatic transport and previously obtained data should be critically revised.

• Klíčová slova
• abiraterone, bioavailability, biodistribution, cinacalcet, lymph duct cannulation, pharmacokinetics,
• MeSH
• aplikace orální MeSH
• biologická dostupnost MeSH
• biologický transport MeSH
• chylomikrony * metabolismus   MeSH
• cykloheximid farmakologie   MeSH
• intestinální absorpce * MeSH
• krysa rodu Rattus MeSH
• léčivé přípravky MeSH
• reprodukovatelnost výsledků MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chylomikrony * MeSH
• cykloheximid MeSH
• léčivé přípravky MeSH

BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.

• Klíčová slova
• Apoptosis, Carbonic anhydrase IX, Chemotherapy, Ectodomain, Hypoxia, Metalloproteinase, Shedding,
• MeSH
• apoptóza účinky léků   genetika   MeSH
• cykloheximid aplikace a dávkování   MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• HeLa buňky MeSH
• hypoxie buňky genetika   MeSH
• karboanhydrasa IX aplikace a dávkování   genetika   metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky aplikace a dávkování   MeSH
• nádory genetika   patologie   MeSH
• protein ADAM17 genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADAM17 protein, human MeSH Prohlížeč
• cykloheximid MeSH
• karboanhydrasa IX MeSH
• monoklonální protilátky MeSH
• protein ADAM17 MeSH

Δ133p53α, a p53 isoform that can inhibit full-length p53, is downregulated at replicative senescence in a manner independent of mRNA regulation and proteasome-mediated degradation. Here we demonstrate that, unlike full-length p53, Δ133p53α is degraded by autophagy during replicative senescence. Pharmacological inhibition of autophagy restores Δ133p53α expression levels in replicatively senescent fibroblasts, without affecting full-length p53. The siRNA-mediated knockdown of pro-autophagic proteins (ATG5, ATG7 and Beclin-1) also restores Δ133p53α expression. The chaperone-associated E3 ubiquitin ligase STUB1, which is known to regulate autophagy, interacts with Δ133p53α and is downregulated at replicative senescence. The siRNA knockdown of STUB1 in proliferating, early-passage fibroblasts induces the autophagic degradation of Δ133p53α and thereby induces senescence. Upon replicative senescence or STUB1 knockdown, Δ133p53α is recruited to autophagosomes, consistent with its autophagic degradation. This study reveals that STUB1 is an endogenous regulator of Δ133p53α degradation and senescence, and identifies a p53 isoform-specific protein turnover mechanism that orchestrates p53-mediated senescence.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• androstadieny farmakologie   MeSH
• autofagie účinky léků   fyziologie   MeSH
• beclin 1 MeSH
• cykloheximid farmakologie   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• genový knockdown MeSH
• kultivované buňky MeSH
• lidé MeSH
• malá interferující RNA MeSH
• membránové proteiny genetika   metabolismus   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• protein - isoformy metabolismus   MeSH
• proteiny asociované s mikrotubuly genetika   metabolismus   MeSH
• proteiny regulující apoptózu genetika   metabolismus   MeSH
• sekvestosom 1 MeSH
• stárnutí buněk fyziologie   MeSH
• ubikvitinligasy genetika   metabolismus   MeSH
• wortmannin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• androstadieny MeSH
• beclin 1 MeSH
• BECN1 protein, human MeSH Prohlížeč
• cykloheximid MeSH
• malá interferující RNA MeSH
• MAP1LC3B protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• nádorový supresorový protein p53 MeSH
• protein - isoformy MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny regulující apoptózu MeSH
• sekvestosom 1 MeSH
• SQSTM1 protein, human MeSH Prohlížeč
• STUB1 protein, human MeSH Prohlížeč
• TP53 protein, human MeSH Prohlížeč
• ubikvitinligasy MeSH
• wortmannin MeSH

Activated hepatic stellate cells (HSC) are a major source offibrous proteins in cirrhotic liver. Inducing or accelerating their apoptosis is a potential way of liver fibrosis treatment. Extracellular matrix (ECM) surrounding cells in tissue affects their differentiation, migration, proliferation and function. Type I collagen is the main ECM component in fibrotic liver. We have examined how this protein modifies apoptosis of normal rat HSC induced by gliotoxin, cycloheximide and cytochalasin D in vitro and spontaneous apoptosis of HSC isolated from CCl4-damaged liver. We have found that type I collagen gel enhances HSC apoptosis regardless of the agent triggering this process.

• MeSH
• apoptóza účinky léků   MeSH
• buněčné kultury MeSH
• chlorid uhličitý MeSH
• cykloheximid MeSH
• cytochalasin D MeSH
• gliotoxin MeSH
• jaterní cirhóza patologie   MeSH
• jaterní hvězdicovité buňky účinky léků   patologie   MeSH
• kolagen typu I farmakologie   MeSH
• krysa rodu Rattus MeSH
• modely nemocí na zvířatech MeSH
• potkani Sprague-Dawley MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorid uhličitý MeSH
• cykloheximid MeSH
• cytochalasin D MeSH
• gliotoxin MeSH
• kolagen typu I MeSH

Small ubiquitin-related modifiers 1, 2 and 3 (SUMO-1, -2, -3), members of the ubiquitin-like protein family, can be conjugated to various cellular proteins. Conjugates of SUMO-2 and SUMO-3 (SUMO-2/3) accumulate in cells exposed to various stress stimuli or to MG132 treatment. Although the proteins modified by SUMO-2/3 during heat shock or under MG132 treatment have been identified, the significance of this modification remains unclear. Our data show that the inhibition of translation by puromycin or cycloheximide blocks both the heat shock and MG132 induced accumulation of SUMO-2/3 conjugates in HEK 293T and U2OS cells. However, the heat shock induced accumulation of SUMO-2/3 conjugates was restored by proteasome inhibition, which suggests that the inhibition of translation did not abolish SUMOylation itself. Furthermore, we show that some of the proteins truncated due to the treatment by low concentration of puromycin are SUMOylated in HEK 293T cells. We suggest that the SUMO-2/3 conjugates accumulating under the heat shock or MG132 treatment result largely from new protein synthesis and that portion of them is incorrectly folded.

• MeSH
• benzochinony farmakologie   MeSH
• biologické modely MeSH
• cykloheximid farmakologie   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• inhibitory proteasomu MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• leupeptiny farmakologie   MeSH
• lidé MeSH
• makrocyklické laktamy farmakologie   MeSH
• malé modifikační proteiny související s ubikvitinem metabolismus   MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• proteosyntéza účinky léků   MeSH
• puromycin farmakologie   MeSH
• reakce na tepelný šok účinky léků   MeSH
• sumoylace účinky léků   MeSH
• ubikvitiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzochinony MeSH
• benzyloxycarbonylleucyl-leucyl-leucine aldehyde MeSH Prohlížeč
• cykloheximid MeSH
• inhibitory proteasomu MeSH
• inhibitory syntézy proteinů MeSH
• leupeptiny MeSH
• makrocyklické laktamy MeSH
• malé modifikační proteiny související s ubikvitinem MeSH
• proteasomový endopeptidasový komplex MeSH
• puromycin MeSH
• SUMO2 protein, human MeSH Prohlížeč
• SUMO3 protein, human MeSH Prohlížeč
• tanespimycin MeSH Prohlížeč
• ubikvitiny MeSH

Quantitative real-time RT-PCR study was conducted to reveal the effects of normal (5 mmol/l) and high (30 mmol/l) glucose without or with oleate (0.3 mmol/l) on mRNA levels of peroxisome proliferator-activated receptor- (PPAR-)alpha, -gamma1, -gamma2, and peroxisome proliferator-activated receptor-gamma coactivator- (PGC-)1alpha and -1beta in commercial human hepatoma-derived HepG2 cells maintained under low-serum condition. Significant decrease in PPAR-gamma1 and PGC-1alpha mRNA levels to about 50 % was observed during the first 4 h incubation period. During the next 4 h period, both PPAR-gamma1 and PGC-1alpha mRNAs were partly but significantly restored in high glucose batches. In this period, the presence of the transcriptional inhibitor actinomycin D revealed a significant protective effect of excess glucose on mature PPAR-gamma1 and PGC-1alpha mRNAs. Furthermore, PPAR-gamma1 and -gamma2 mRNAs were differentially superinduced 1.2-2.5 fold in cells upon the administration of the translational inhibitor cycloheximide. When the cells were co-treated with the combination of cycloheximide and actinomycin D, superinduction was completely suppressed, however. Altogether, the experiments revealed, first, an unexpected protective effect of abundant glucose on PPAR-gamma1 and PGC-1alpha mRNAs in HepG2 cells. Second, we demonstrated cycloheximide-induced, transcription-dependent upregulation of mature PPAR-gamma1 and -gamma2 mRNAs in HepG2 cells associated with preferential expression of the PPAR-gamma2 mRNA variant. The results draw attention to as yet unexplored mechanisms involved in the control of PPAR and PGC genes.

• MeSH
• buňky Hep G2 MeSH
• cykloheximid farmakologie   MeSH
• genetická variace MeSH
• glukosa farmakologie   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• kyselina olejová farmakologie   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• nádorové buněčné linie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• PPAR gama genetika   metabolismus   MeSH
• regulace genové exprese účinky léků   MeSH
• upregulace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cykloheximid MeSH
• glukosa MeSH
• inhibitory syntézy proteinů MeSH
• kyselina olejová MeSH
• messenger RNA MeSH
• PPAR gama MeSH

Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2alpha/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Deltalsm4DeltaC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2alpha became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2alpha. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.

• MeSH
• cykloheximid farmakologie   MeSH
• cytoplazmatická granula účinky léků   metabolismus   fyziologie   MeSH
• down regulace fyziologie   MeSH
• eukaryotický iniciační faktor 2 metabolismus   MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• fosforylace fyziologie   MeSH
• geneticky modifikované organismy MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• protein-serin-threoninkinasy fyziologie   MeSH
• proteosyntéza účinky léků   fyziologie   MeSH
• reakce na tepelný šok účinky léků   fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   fyziologie   MeSH
• tkáňová distribuce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cykloheximid MeSH
• eukaryotický iniciační faktor 2 MeSH
• eukaryotický iniciační faktor 3 MeSH
• GCN2 protein, S cerevisiae MeSH Prohlížeč
• inhibitory syntézy proteinů MeSH
• protein-serin-threoninkinasy MeSH
• Saccharomyces cerevisiae - proteiny MeSH

Loss of programmed cell death pathways is one of the features of malignancy that complicate the response of cancer cells to a therapy. Activation of alternative cell death pathways offers a promising approach to enhance efficiency of cancer chemotherapy. We analysed programmed cell death pathways of v-myb-transformed BM2 monoblasts induced by arsenic trioxide, cycloheximide and camptothecin with U937 promonocytes as a reference cell line. We show that induced death of BM2 cells is not executed by caspases but rather by alternative cell death pathways. Camptothecin induces the lysosome-dependent cell death, arsenic trioxide induces autophagy, and most of cycloheximide-treated BM2 cells die by necrosis. The fact that alternative cell death pathways can be switched in cells with defects in activation and/or function of caspases suggests that understanding and targeting of these pathways could improve therapy of cancer cells suffering from defective apoptosis.

• MeSH
• antitumorózní látky farmakologie   MeSH
• apoptóza účinky léků   MeSH
• arsenikové přípravky farmakologie   MeSH
• autofagie účinky léků   MeSH
• cykloheximid farmakologie   MeSH
• fluorescenční mikroskopie MeSH
• geny myb fyziologie   MeSH
• kamptothecin farmakologie   MeSH
• kaspasy metabolismus   MeSH
• kur domácí MeSH
• lidé MeSH
• nádorová transformace buněk patologie   MeSH
• nekróza MeSH
• oxid arsenitý MeSH
• oxidy farmakologie   MeSH
• signální transdukce účinky léků   MeSH
• transformované buněčné linie MeSH
• U937 buňky účinky léků   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky MeSH
• arsenikové přípravky MeSH
• cykloheximid MeSH
• kamptothecin MeSH
• kaspasy MeSH
• oxid arsenitý MeSH
• oxidy MeSH

Wnt signaling enhances cell proliferation and the maintenance of hematopoietic cells. In contrast, cytotoxic ligand Apo2L/TRAIL induces the apoptosis of various transformed cells. We observed that co-culture of human pre-B leukemia cells KM3 and REH with Wnt1- or Wnt3a-producing rat embryonic fibroblasts efficiently suppressed Apo2L/TRAIL-induced apoptosis of the lymphoid cells. This suppression occurs at the early stages of the Apo2L/TRAIL apoptotic cascade and, interestingly, the activation of the Wnt pathway alone in human leukemia cells is not sufficient for their full anti-apoptotic protection. We hypothesize that a stimulus emanating specifically from Wnt1- or Wnt3a-expressing rat fibroblasts is responsible for the observed resistance to Apo2L/TRAIL. This anti-apoptotic signaling was significantly hampered by the inhibition of the MEK1/ERK1/2 or NFkappaB pathways in KM3 and REH cells. Our results imply that paracrine Wnt-related signals could be important for the survival of pre-B cell-derived malignancies.

• MeSH
• apoptóza fyziologie   MeSH
• beta-katenin fyziologie   MeSH
• cykloheximid farmakologie   MeSH
• daktinomycin farmakologie   MeSH
• kokultivační techniky MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• pre-B-buněčná leukemie MeSH
• protein TRAIL antagonisté a inhibitory   MeSH
• protein Wnt1 biosyntéza   MeSH
• protein Wnt3 MeSH
• protein Wnt3A MeSH
• proteiny regulující apoptózu fyziologie   MeSH
• proteiny Wnt biosyntéza   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin MeSH
• cykloheximid MeSH
• daktinomycin MeSH
• protein TRAIL MeSH
• protein Wnt1 MeSH
• protein Wnt3 MeSH
• protein Wnt3A MeSH
• proteiny regulující apoptózu MeSH
• proteiny Wnt MeSH
• Wnt1 protein, rat MeSH Prohlížeč
• WNT3A protein, human MeSH Prohlížeč

AIMS: Very modest changes in mRNA stability can affect critical points in cellular energy pathways. The aim of this study was to investigate the impact of energy abundant substrates on peroxisome proliferator-activated receptors (PPARs) and PPAR-gamma coactivators (PGCs) mRNA's steady-state levels. METHODS: Quantitative RT-PCR study was performed to assess the effect of zero or normal (5 mmol/l) glucose and/or oleic acid (0.3 mmol/l) on mRNA levels of (PPARs) (PGCs) in HepG2 cells. RESULTS: PGC-1alpha mRNA was significantly upregulated in glucose deprived cells (123 % of the control level; p < 0.05), while PGC-1beta mRNA was significantly enhanced in oleate-fed cells (134 % and 160 % of control levels for zero glucose plus oleate and normal glucose plus oleate, respectively; p < 0.05) during the 0.5 h incubation. Upon the 4 h incubation, PPAR-gamma1 and PGC-1alpha mRNAs were significantly elevated in cells lacking glucose (142 % and 163 % of control levels, respectively; p < 0.05). Oleate significantly suppressed PPAR-alpha and PGC-1beta mRNA levels in glucose-deprived cells (58 % and 49 % of control levels, respectively; p < 0.05). PPAR-gamma1 and -gamma2 mRNAs were significantly superinduced when the cells were treated with cycloheximide, whereas PPAR-alpha and PGC-1alpha and-1beta mRNAs were destabilized. Upon actinomycin D treatment, glucose shortage significantly stabilized PPAR-alpha mRNA, while PGC-1alpha mRNA was destabilized by oleate in glucose-deprived cells. CONCLUSIONS: Our findings provide evidence that transcriptional processes that are under the control of energetic substrates are interconnected with concurrent translational processes that can change stability of mRNAs.

• MeSH
• cykloheximid farmakologie   MeSH
• daktinomycin farmakologie   MeSH
• glukosa metabolismus   farmakologie   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• kyselina olejová farmakologie   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• nádorové buněčné linie metabolismus   MeSH
• nádory jater metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• PPAR gama genetika   metabolismus   MeSH
• receptory aktivované proliferátory peroxizomů genetika   metabolismus   MeSH
• techniky in vitro MeSH
• upregulace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cykloheximid MeSH
• daktinomycin MeSH
• glukosa MeSH
• inhibitory syntézy proteinů MeSH
• kyselina olejová MeSH
• messenger RNA MeSH
• PPAR gama MeSH
• receptory aktivované proliferátory peroxizomů MeSH