Activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (AKT) pathway is associated with three major radioresistance mechanisms: intrinsic radioresistance; tumour-cell proliferation; and hypoxia. Monitoring and manipulation of this signal-transduction pathway can have important implications for the management of head and neck cancer, because activation of the PI3-K/AKT pathway is a frequent event in these tumours. PI3-K/AKT signalling regulates cellular processes, including proliferation, invasion, apoptosis, and the upregulation of hypoxia-related proteins. Activation of this pathway can be caused by stimulation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). In clinical trials, a strong and independent association has been noted between expression of activated AKT and treatment outcome. Therefore, the search for molecular predictors of sensitivity to EGFR-directed treatment should be extended to markers of PI3-K/AKT activation. Another strategy might be the direct targeting and inhibition of this pathway. Such inhibition will enhance the efficacy of radiotherapy, by antagonising radiation-induced cellular defense mechanisms, especially in tumours that have activated the PI3-K/AKT cascade. Thus, the activation status of this pathway might be a key element for the prediction of treatment response and for therapeutic targeting in head and neck cancer.

• MeSH
• aktivace enzymů účinky záření   MeSH
• fosfatidylinositol-3-kinasy metabolismus   účinky záření   MeSH
• hypoxie buňky účinky záření   MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• lidé MeSH
• nádory hlavy a krku enzymologie   radioterapie   MeSH
• prognóza MeSH
• protoonkogenní proteiny c-akt antagonisté a inhibitory   metabolismus   účinky záření   MeSH
• signální transdukce fyziologie   účinky záření   MeSH
• spinocelulární karcinom enzymologie   radioterapie   MeSH
• Check Tag
• lidé MeSH

Akt kinase regulates numerous cell functions including glucose metabolism, cell growth, survival, protein synthesis, and control of local hemodynamics. mTOR is one of down-stream effectors of Akt involved in the initiation of protein translation. However, renal Akt signaling in Type 1 diabetes (DM) in vivo, in particular under the conditions reflecting differences in metabolic control, has received less attention. Renal cortical activity and expression of Akt and mTOR (kinase assay, western blotting) were determined in streptozotocin-diabetic rats (D) with different levels of glycemic control (blood glucose 22.0±1.0, 13.4±1.5, 8.1±0.4 mmol/l, p<0.05 between the groups), achieved by varying insulin treatment (0, 4 and 12 IU/day), and in control rats with (C4) or without (C) chronic insulin administration. Renal Akt activity was reduced in D rats without insulin treatment and severe hyperglycemia (D-0, -62 %, p<0.01 vs. C), partially restored in moderately hyperglycemic rats (D-4, -30 %, p<0.05 vs. C), and normalized in D rats with intensive insulin and tight metabolic control (D-12). Expression of active mTOR paralleled Akt activity in D-0 (-51 %, p<0.01 vs. C), but not in D-4 and D- 12 that demonstrated increases in active mTOR (+55 %, +80 % resp., p<0.05) as compared to C. Moreover, insulin activated renal Akt (+82 %, p<0.01), but not mTOR in C4. In conclusion, glycemic control and intensity of insulin treatment are important modulators of renal Akt and mTOR activity in diabetes. While Akt activity is reversible by tight metabolic control, combination of hyperglycemia and insulin treatment resulted in enhancement of mTOR activity. In addition to Akt, other signaling pathways likely contribute to regulation of renal mTOR activity in diabetes.

• Klíčová slova
• Akt kinase, Diabetic nephropathy, Insulin, Mammalian targetof rapmycin, mTOR,
• MeSH
• diabetes mellitus 1. typu enzymologie   farmakoterapie   komplikace   MeSH
• diabetické nefropatie enzymologie   etiologie   farmakoterapie   MeSH
• experimentální diabetes mellitus enzymologie   farmakoterapie   komplikace   MeSH
• financování organizované MeSH
• fosforylace MeSH
• hypoglykemika terapeutické užití   MeSH
• inzulin krev   terapeutické užití   MeSH
• kinasa 3 glykogensynthasy metabolismus   MeSH
• krevní glukóza metabolismus   MeSH
• krysa rodu rattus MeSH
• kůra ledviny enzymologie   účinky léků   MeSH
• proteinkinasy metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• signální transdukce MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

• MeSH
• antitumorózní látky farmakokinetika   MeSH
• lidé MeSH
• nádory patofyziologie   MeSH
• proteinkinasy MeSH
• sirolimus farmakokinetika   MeSH
• Check Tag
• lidé MeSH

Insulin resistance (IR) and consequent hyperinsulinemia are hallmarks of Type 2 diabetes (DM2). Akt kinase (Akt) is an important molecule in insulin signaling, implicated in regulation of glucose uptake, cell growth, cell survival, protein synthesis, and endothelial nitric oxide (NO) production. Impaired Akt activation in insulin-sensitive tissues contributes to IR. However, Akt activity in other tissues, particularly those affected by complications of DM2, has been less studied. We hypothesized that hyperinsulinemia could have an impact on activity of Akt and its effectors involved in regulation of renal morphology and function in DM2. To address this issue, renal cortical Akt was determined in obese Zucker rats (ZO), a model of DM2, and lean controls (ZL). We also studied expression and phosphorylation of the mammalian target of rapamycin (mTOR) and endothelial NO synthase (eNOS), molecules downstream of Akt in the insulin signaling cascade, and documented modulators of renal injury. Akt activity was measured by a kinase assay with GSK-3 as a substrate. Expression of phosphorylated (active) and total proteins was measured by immunoblotting and immunohistochemistry. Renal Akt activity was increased in ZO as compared to ZL rats, in parallel with progressive hyperinsulinemia. No differences in Akt were observed in the skeletal muscle. Corresponding to increases in Akt activity, ZO rats demonstrated enhanced phosphorylation of renal mTOR. Acute PI3K inhibition with wortmannin (100 mug/kg) attenuated renal Akt and mTOR activities in ZO, but not in ZL rats. In contrast to mTOR, eNOS phosphorylation was similar in ZO and ZL rats, despite higher total eNOS expression. In conclusion, ZO rats demonstrated increases in renal Akt and mTOR activity and expression. However, eNOS phosphorylation did not follow this pattern. These data suggest that DM2 is associated with selective IR in the kidney, allowing pro-growth signaling via mTOR, whereas potentially protective effects mediated by eNOS are blunted.

• Klíčová slova
• Phosphatidylinositol 3-Kinases,
• MeSH
• androstadieny farmakologie   MeSH
• diabetes mellitus 2. typu metabolismus   MeSH
• financování organizované MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem antagonisté a inhibitory   MeSH
• hyperinzulinismus metabolismus   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• inzulin krev   MeSH
• inzulinová rezistence fyziologie   MeSH
• krysa rodu rattus MeSH
• ledviny enzymologie   MeSH
• modely nemocí na zvířatech MeSH
• obezita metabolismus   MeSH
• potkani Zucker MeSH
• proteinkinasy metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• synthasa oxidu dusnatého, typ III metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčné dýchání účinky léků   MeSH
• down regulace účinky léků   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• fosforylace účinky léků   MeSH
• glukosa metabolismus   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• kyselina mléčná biosyntéza   MeSH
• lidé MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• molybden farmakologie   MeSH
• myši inbrední C57BL MeSH
• nádorové buněčné linie MeSH
• neuroblastom enzymologie   metabolismus   patologie   MeSH
• neurony účinky léků   metabolismus   MeSH
• oxidativní fosforylace účinky léků   MeSH
• protoonkogenní proteiny c-akt antagonisté a inhibitory   metabolismus   MeSH
• spotřeba kyslíku účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 microM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.

• MeSH
• aktivace enzymů účinky léků   fyziologie   MeSH
• buněčné kultury MeSH
• dibutyryl cyklický AMP farmakologie   MeSH
• fosfatidylinositoly farmakologie   MeSH
• fosforylace účinky léků   MeSH
• inhibitory enzymů farmakologie   MeSH
• kultivované buňky MeSH
• meióza fyziologie   MeSH
• onkogenní protein v-akt antagonisté a inhibitory   metabolismus   fyziologie   MeSH
• oocyty účinky léků   metabolismus   fyziologie   MeSH
• oogeneze účinky léků   fyziologie   MeSH
• prasata metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We examined the involvement of phosphatidylinositol 3-kinase (PI3K) and its effector protein kinase B (Akt) in cardioprotective effects of ischemic preconditioning (PC) with particular regards to its role in the protection against ischemia-induced arrhythmias in isolated perfused rat heart. PI3K/Akt inhibitor wortmannin (100 nM) was administered 15 min prior to 30-min regional (left anterior descending coronary artery occlusion) ischemia for the study of ischemic arrhythmias in the hearts perfused at constant coronary flow or prior to 30-min global ischemia followed by 2-h reperfusion for the infarct size (IS) determination (tetrazolium staining) in the hearts perfused at constant pressure. PC procedure (one cycle of ischemia/reperfusion, 5 min each) significantly reduced the total number of ventricular premature complexes (PVC) and severity of arrhythmias (arrhythmia score; AS) over the whole period of left anterior descending coronary artery occlusion in comparison with non-PC controls (PVC 166+/-40; AS 1.6+/-0.2 vs. 550+/-60 and 3.2+/-0.2; respectively; P<0.05). In a setting of global ischemia/reperfusion, PC decreased IS (in % of the left ventricle, LV) by 73 %. Pretreatment with wortmannin modified neither arrhythmogenesis nor IS in the non-PC hearts. Bracketing of PC with wortmannin did not abolish antiarrhythmic protection (PVC 92+/-25; AS 1.7+/-0.2; P<0.05 vs. non-PC hearts). On the other hand, wortmannin increased IS/LV in the PC hearts to 24+/-1.2 % as compared with 9 +/- 0.6 % in the untreated ones (P<0.05). In conclusion, PI3K/Akt inhibition did not affect reduced arrhythmogenesis during ischemia in the PC hearts indicating that in contrast to its positive role in the irreversible myocardial injury, PI3K/Akt activity is not required for protection induced by PC against ischemic arrhythmias in the rat heart.

• MeSH
• androstadieny farmakologie   MeSH
• financování organizované MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• infarkt myokardu enzymologie   etiologie   prevence a kontrola   MeSH
• inhibitory fosfoinositid-3-kinasy MeSH
• inhibitory proteinkinas farmakologie   MeSH
• ischemická choroba srdeční enzymologie   komplikace   terapie   MeSH
• ischemické přivykání MeSH
• krysa rodu rattus MeSH
• myokard enzymologie   patologie   MeSH
• perfuze MeSH
• potkani Wistar MeSH
• protoonkogenní proteiny c-akt antagonisté a inhibitory   metabolismus   MeSH
• reperfuzní poškození myokardu enzymologie   etiologie   prevence a kontrola   MeSH
• srdeční arytmie enzymologie   etiologie   prevence a kontrola   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

BACKGROUND: PTEN is a tumour suppressor protein with phosphatase activity frequently altered in several types of human cancers. MATERIALS AND METHODS: The PTEN effect was studied on the cell cycle (by bromdeoxyuridine incorporation) and on the phosphatidylinositol-3-kinase/protein kinase B/Akt (PI3-K/PKB/Akt) pathway regulating proteins (by immunocytochemical, Western blot analysis and kinase assay) upon transfection of wild-type PTEN and its mutant H123Y in breast cancer cell lines. RESULTS: The expression of the important proteins in the MCF-7 and BT-549 cells was characterised and the cellular localisation of PTEN was analysed. Transfection of H123Y led to the down-regulation of p27(Kip1) and p21(Waf1/Cip1) protein levels and the up-regulation of phosphorylated PKB/Akt. An overexpression of PTEN decreased cyclin E/cdk2 activity and inhibited S-phase entry in MCF-7. In BT-549 these changes were not observed, but overexpression of PTEN led to a diminution of PKB/Akt phosphorylation. CONCLUSION: PTEN function is mediated through the inhibition of the cell cycle and PKB/Akt phosphorylation in breast cancer cells.

• MeSH
• buněčný cyklus fyziologie   MeSH
• cyklin-dependentní kinasa 2 metabolismus   MeSH
• cykliny biosyntéza   MeSH
• financování organizované MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosfohydroláza PTEN biosyntéza   genetika   metabolismus   MeSH
• fosforylace MeSH
• inhibitor p21 cyklin-dependentní kinasy biosyntéza   MeSH
• intracelulární signální peptidy a proteiny MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prsu enzymologie   genetika   metabolismus   patologie   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• S fáze genetika   MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH

BACKGROUND: Interleukin-22 (IL-22) is a pro-inflammatory cytokine released during the immune response in chronic liver injury. Although IL-22 mediates tissue regeneration, its uncontrolled production may generate a carcinogenic environment resulting in hepatocellular carcinoma (HCC). This study aims to identify the effect of IL-22 on anti-apoptotic and metastatic genes and the molecular pathways responsible for IL-22-mediated hepatic carcinogenesis. METHODS AND RESULTS: Three cancerous liver lines, HepG2, SNU-387, Huh7, and one normal liver line, THLE2, were treated with IL-22. RT-qPCR analysis was conducted to study the role of IL-22 in altering the expression levels of anti-apoptotic genes, MCL-1 and BCL-2, and metastatic genes, MMP-7 and MMP-9. A significant increase in expression levels of these genes was observed after IL-22 treatment. Furthermore, to explore the major pathways involved in IL-22-mediated upregulation of anti-apoptotic and metastatic genes, cells were treated with inhibitors of JAK/STAT and PI3K/AKT pathways along with IL-22. Resultantly, a significant decrease in expression levels of target genes was observed, indicating the involvement of JAK/STAT and PI3K/AKT signaling cascades in IL-22-mediated oncogenesis. Finally, Cell Scratch assay was performed to check the effect of IL-22 and inhibitors of JAK/STAT and PI3K/AKT on the metastatic potential of liver cells. While migration was observed in Huh7 and THLE2 cells treated with IL-22, no migration was observed in cells treated with IL-22 along with JAK/STAT and PI3K/AKT inhibitors. Results indicate that IL-22 encourages metastasis in HCC cells via the JAK/STAT and PI3K/AKT pathways. CONCLUSION: Results showed that IL-22 upregulates anti-apoptotic and metastatic genes in HCC through JAK/STAT and PI3K/AKT signaling pathways.

• MeSH
• 1-fosfatidylinositol-3-kinasa metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• hepatocelulární karcinom * metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory jater * metabolismus   MeSH
• proliferace buněk genetika   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase conserved in all eukaryotes that plays a key role in cell growth and is a central effector of several pathways regulating essential cell functions. Hyperactivation of the mTORdependent signalling pathway occurs in many human diseases and may be a selective target for their therapy. However, the dual nature of mTOR, existing in two multiprotein complexes mTORC1 and mTORC2 driven by different feedback loops, decreases the therapeutic effects of rapamycin, the specific mTOR inhibitor. In the present study we demonstrate that the mTORC1 signalling pathway is highly activated in human melanoma cells and that up-regulation of this pathway along with the growth and malignity of these cells could be suppressed by disruption of the Src activity. SU6656, the selective inhibitor of the Src kinase activity, decreased up-regulation of the mTORC1 signalling and moreover, unlike rapamycin, it did not induce the activation of Akt/PKB and its downstream targets in HBL melanoma cells. The Src protein was found to be associated with raptor in the mTORC1 complex immunoprecipitated from these cells, suggesting that the Src activity might be a new attractive target for monotherapeutic inhibition of the up-regulated mTORC1 signalling pathway.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aktivace enzymů účinky léků   MeSH
• fosforylace účinky léků   MeSH
• indoly farmakologie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• melanom enzymologie   patologie   MeSH
• multiproteinové komplexy antagonisté a inhibitory   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• sirolimus farmakologie   MeSH
• skupina kinas odvozených od src-genu antagonisté a inhibitory   metabolismus   MeSH
• sulfonamidy farmakologie   MeSH
• testy nádorových kmenových buněk MeSH
• TOR serin-threoninkinasy antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH