u-0126 and thiazolyl-blue

Chronic myelogenous leukemia (CML) is triggered by the constitutively activated BCR-ABL oncoprotein and multiple downstream signaling pathways, including the Raf/MEK/ERK, Akt/mTOR, SRC, and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for CML. However, the development of imatinib resistance has become a new challenge for CML treatment. Here, we investigated the expression levels of the signaling pathways to explore the cause of imatinib resistance and seek new reversing drugs. Our results showed that abnormal activation of the BCR-ABL-independent Lyn/ERK signaling pathway was involved in imatinib-resistance of K562R cells. Furthermore, p-Lyn and p-ERK were up-regulated after treatment with imatinib alone. However, U0126, a MEK1/2 inhibitor, could counteract the up-regulation induced by imatinib, and the combination of imatinib and U0126 could overcome the resistance to imatinib in K562R cells. In conclusion, our studies suggest that the combination of imatinib and an inhibitor of the ERK signaling pathway may be effective in imatinib-resistant CML patients.

Topics: Antineoplastic Agents; Benzamides; Blotting, Western; Butadienes; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Flow Cytometry; Humans; Imatinib Mesylate; Indicators and Reagents; K562 Cells; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Pyrimidines; Real-Time Polymerase Chain Reaction; src-Family Kinases; STAT5 Transcription Factor; Tetrazolium Salts; Thiazoles; TOR Serine-Threonine Kinases

FGF-2 stimulates cell proliferation of rabbit corneal endothelial cells (rCECs) by degrading the cyclin-dependent kinase inhibitor p27(Kip1) (p27) through its phosphorylation mechanism. The authors investigated whether the cell proliferation of human CECs (hCECs) is also induced by FGF-2 stimulation through the p27 phosphorylation pathway.. Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Transfection of hCECs with small interference RNA (siRNA) was performed using a transfection reagent.. FGF-2 stimulated cell proliferation in hCECs; the FGF-2 action was completely blocked by pathway-specific inhibitors for PI 3-kinase (LY294002) and MEK1/2 (U0126), respectively. Using immunoblotting, the authors showed that FGF-2 induced phosphorylation of p27 at both serine 10 (Ser10) and threonine 187 (Thr187) sites. These effects were also completely blocked by LY294002 or U0126. The authors then determined cross-talk between PI 3-kinase and extracellular signal-regulated kinase (ERK)1/2; blocking of ERK1/2 activation by LY294002 indicated that in hCECs ERK1/2 works as a downstream effector to PI 3-kinase for cell proliferation induced by FGF-2, whereas the ERK1/2 pathway in rCECs is parallel to the PI 3-kinase pathway. However, the downstream mechanism involved in cell cycle progression in hCECs is identical to that of rCECs: phosphorylation of p27 at Ser10 was mediated by kinase-interacting stathmin (KIS), confirmed with siRNA to KIS, and phosphorylation of p27 at Thr187 was mediated by cell division cycle 25A (Cdc25A), confirmed using Cdc25A inhibitor. CONCLUSIONS; FGF-2 stimulates proliferation of hCECs through PI 3-kinase and its downstream target ERK1/2 pathways. This linear signal transduction significantly downregulates p27 through its phosphorylation at both Ser10 and Thr187 sites mediated by KIS and Cdc25A, respectively.

Topics: Adolescent; Adult; Aged; Butadienes; Cell Proliferation; Chromones; Cyclin-Dependent Kinase Inhibitor p27; Endothelium, Corneal; Enzyme Inhibitors; Fibroblast Growth Factor 2; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Microscopy, Confocal; Middle Aged; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; RNA, Small Interfering; Serine; Tetrazolium Salts; Thiazoles; Threonine; Tissue Donors; Transfection; Young Adult

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.

Topics: Analysis of Variance; Animals; Blotting, Western; Bromodeoxyuridine; Butadienes; Caspase 3; Cell Count; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression; Glial Fibrillary Acidic Protein; Glioma; Immunohistochemistry; Mice; Nitriles; Tetrazolium Salts; Thiazoles

We have recently reported that the ASK1-p38 MAPK pathway has an important role in the low potassium (LK)-induced apoptosis of cultured cerebellar granule neurons. In the present study, we observed that ERK1/2 were significantly activated 6 h after a change of medium from HK (high potassium) to LK. In addition, U0126, a specific inhibitor of MEKs, remarkably prevented the apoptosis of cultured cerebellar granule neurons. Then, we examined the mechanism underlying the activation of ERK1/2 in the LK-induced apoptotic pathway. The addition of SB203580, an inhibitor of p38 MAPK, suppressed the increase in the phosphorylation of ERK1/2 after the change to LK medium. Furthermore, we found that the expression of a constitutively active mutant of ASK1, an upstream kinase of p38 MAPK, enhanced the phosphorylation of ERK1/2. These results suggest that ERK1/2 play a crucial role in LK-induced apoptosis of cultured cerebellar granule neurons and that the LK-stimulated activation of ERK1/2 is regulated by the ASK1-p38 MAPK pathway.

Topics: Adenoviridae; Animals; Apoptosis; Blotting, Western; Butadienes; Cells, Cultured; Cerebellum; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Immunohistochemistry; Male; MAP Kinase Kinase Kinase 5; Mitogen-Activated Protein Kinase 7; Neurons; Nitriles; p38 Mitogen-Activated Protein Kinases; Potassium Deficiency; Rats; Rats, Wistar; Signal Transduction; Tetrazolium Salts; Thiazoles

Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attempt to further elucidate the signal transduction pathways involved in the cell death/cell survival associated with excitotoxicity, we have used an in vivo model of excitotoxicity employing kainic acid (KA)-induced neurotoxicity. Here, we show that extracellular signal-related kinase (ERK) 2, but not ERK 1, is phosphorylated and thereby activated in the hippocampus and cerebellum of kainic acid-treated mice. Phosphorylation and hence inactivation of glycogen synthase kinase 3beta (GSK-3beta), a general survival factor, is often a downstream consequence of mitogen-activated protein kinase pathway activation. Indeed, GSK-3beta phosphorylation occurred in response to kainic acid exclusively in the affected hippocampus, but not as a consequence of ERK activation. This may represent a compensatory attempt at self-protection by the cells in this particular brain region. A role for GSK-3beta inhibition in cell survival was further supported by the fact that pharmacological inhibition of GSK-3beta using lithium chloride was protective against kainic acid-induced excitotoxicity in hippocampal slice cultures. This work supports a role for GSK-3beta in cell death in response to excitotoxins in vivo and further confirms that GSK-3beta plays a role in cell death/cell survival pathways.

Topics: Animals; Behavior, Animal; Blotting, Western; Butadienes; Cell Death; Cell Survival; Cerebellum; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Immunohistochemistry; Kainic Acid; Lithium Chloride; Male; Mice; Mitogen-Activated Protein Kinase 1; Neurotoxicity Syndromes; Nitriles; Organ Culture Techniques; Phosphorylation; Serine; Tetrazolium Salts; Thiazoles; Time Factors; Tyrosine

Neurotrophic factors have been proposed for the treatment of a variety of neurological diseases. However, to this point they have failed in clinical trials. One potential problem is that while neurotrophic factors attenuate apoptosis, they have the potential to enhance necrosis. In this study we show that neurotrophin-4 (NT-4) attenuated apoptotic neuronal death while potentiating necrotic neuronal death in cortical cultures. The protective effects of NT-4 were not blocked by the mitogen-activated protein kinase kinase (MEK) inhibitors PD098059 or U0126, while the injury potentiation by NT-4 was blocked by these inhibitors. NT-4 stimulated the phosphorylation of ERK1/2 and this phosphorylation was attenuated by U0126 and PD098059. The results indicate a disassociation between the pathway by which NT-4 potentiates necrosis, and that by which it attenuates apoptosis, and suggest that addition of a MEK inhibitor may enhance the beneficial effects of NT-4 in treating complex injuries such as occur in vivo.

Topics: Animals; Butadienes; Cell Death; Cells, Cultured; Cyclosporine; Enzyme Activation; Flavonoids; Immunosuppressive Agents; L-Lactate Dehydrogenase; Mice; Mitogen-Activated Protein Kinases; Necrosis; Nerve Growth Factors; Neurons; Nitriles; Phosphorylation; Tetrazolium Salts; Thiazoles

Endogenous cardiotonic steroids (ECS) are putative ligands of the inhibitory binding site of the membrane sodium pump (Na+, K+-ATPase). There is growing evidence that cardiotonic steroids may promote the growth of cardiac and vascular myocytes, including evidence indicating growth stimulation at concentrations in the same range as circulating ECS concentrations. We investigated four parameters to determine whether ouabain, a proposed ECS, promotes growth of immortalized rat proximal tubule epithelial cells: cell count by hemocytometer; metabolic activity as reflected in the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its formazan product (MA); DNA synthesis reflected as bromodeoxyuridine incorporation (DNA); and mitosis reflected as histone phosphorylation state detected using anti-phosphohistone 3 antibody (HP). Maximum stimulatory responses were observed at 1 nm ouabain (MA, 20.3% increase, p < 0.01; DNA, 28.4% increase, p < 0.001; HP, maximum response at 0.5 h, 50% increase, p < 0.001). We observed that growth stimulation was associated with stimulation of ERK1/2 phosphorylation (ERK-P), and both growth and ERK-P could be blocked by the MEK inhibitor (U0126, 100 nm). Western blot analysis revealed that the only alpha isoform of Na+, K+-ATPase that could be detected in these cultures was the highly ouabain-resistant alpha1 isoform. Measurement of ouabain inhibition of ion transport in these cultures using 86Rb+ uptake revealed the predominance of the expected ouabain-resistant isoform (IC50 = 24 microm) and an additional minor ( approximately 15%) ouabain-sensitive inhibition with IC50 approximately 30 pm. Similar bimodal transport inhibition curves were obtained in freshly dissected rat proximal tubules. These results indicate that renal epithelial cells may be a sensitive target of the ERK1/2-activating and growth-promoting effects of ouabain even in the presence of ouabain-resistant Na+, K+-ATPase.

Topics: Animals; Biological Transport; Blotting, Western; Bromodeoxyuridine; Butadienes; Cell Division; Cells, Cultured; Coloring Agents; DNA; Dose-Response Relationship, Drug; Drug Resistance; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Histones; Inhibitory Concentration 50; Ions; Kidney; Male; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Ouabain; Phosphorylation; Protein Isoforms; Rats; Regression Analysis; Signal Transduction; Sodium; Sodium-Potassium-Exchanging ATPase; Tetrazolium Salts; Thiazoles; Time Factors

Insoluble alpha-synuclein accumulates in Parkinson's disease, diffuse Lewy body disease, and multiple system atrophy. However, the relationship between its accumulation and pathogenesis is still unclear. Recently, we reported that overexpression of alpha-synuclein affects Elk-1 phosphorylation in cultured cells, which is mainly performed by mitogen-activated protein kinases (MAPKs). We further examined the relationship between MAPK signaling and the effects of alpha-synuclein expression on ecdysone-inducible neuro2a cell lines and found that cells expressing alpha-synuclein had less phosphorylated MAPKs. Moreover, they showed significant cell death when the concentration of serum in the culture medium was reduced. Under normal serum conditions, the addition of the MAPK inhibitor U0126 also caused cell death in alpha-synuclein-expressing cells. Transfection of constitutively active MEK-1 resulted in MAPK phosphorylation in alpha-synuclein-expressing cells and improved cell viability even under reduced serum conditions. Thus, we conclude that alpha-synuclein regulates the MAPK pathway by reducing the amount of available active MAPK. Our findings suggest a mechanism for pathogenesis and thus offer therapeutic insight into synucleinopathies.

Topics: 14-3-3 Proteins; alpha-Synuclein; Amino Acid Sequence; Apoptosis; Blotting, Northern; Butadienes; Cell Death; Cell Line; Cell Survival; Coloring Agents; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Library; Glutathione Transferase; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; MAP Kinase Signaling System; Molecular Sequence Data; Mutation; Nerve Tissue Proteins; Nitriles; Phosphorylation; Plasmids; Precipitin Tests; Protein Binding; Recombinant Fusion Proteins; Sequence Homology, Amino Acid; Signal Transduction; Synucleins; Tetrazolium Salts; Thiazoles; Time Factors; Transfection; Tyrosine 3-Monooxygenase