u-0126 and Necrosis

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2'-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34, 4'-OH PCB 35, 4-OH PCB 36, 4'-OH PCB 36, 4-OH PCB 39, and 4'-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14<4-OH PCB 36<4-OH PCB 34<4'-OH PCB 36<4'-OH PCB 68<4-OH PCB 39<4'-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds.

Topics: Animals; Animals, Newborn; Antioxidants; Apoptosis; Biotransformation; Butadienes; Cells, Cultured; Cerebellum; Dose-Response Relationship, Drug; Environmental Pollutants; Hydroxylation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Necrosis; Nitriles; Oxidative Stress; Polychlorinated Biphenyls; Protein Kinase Inhibitors; Rats; Reactive Oxygen Species; Time Factors; Vitamin E

BW 5147 (murine lymphoma cell line). We analyzed the effect of H2O2 in cell proliferation testing nitric oxide and apoptosis. Enzymes involved in the regulation of H2O2 levels as superoxide dismutase (SOD) and peroxidase (PER) were analyzed. H2O2 exerted a biphasic effect. The inhibitory effect of H2O2 was related to the activation of the ERK and P38 pathway, NO production and apoptosis. The high proliferation was associated with a low level of H2O2 related to a low SOD and a high PER activities. Drugs capable of producing an increase in H2O2 levels could be used in cancer.

Topics: Animals; Apoptosis; Butadienes; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Hydrogen Peroxide; Imidazoles; Lymph Nodes; Lymphocytes; Lymphoma, T-Cell; Male; Mice; Mice, Inbred C3H; Mitogen-Activated Protein Kinase Kinases; Necrosis; NF-kappa B; Nitric Oxide; Nitriles; Nitrites; omega-N-Methylarginine; p38 Mitogen-Activated Protein Kinases; Peroxidase; Protein Kinase Inhibitors; Pyridines; Superoxide Dismutase; Superoxides

Dysfunctional dendritic cells (DC) are common in cancer patients; however, the underlying molecular targets are poorly understood. Nevertheless, adoptive-transfer and in situ vaccination protocols continue, largely without addressing immune-suppression. Understanding tumour-mediated DC suppression would assist rational design of next generation immunotherapy. This study used a tumour-lysate loaded DC model of adoptive immunotherapy and also necrotic cells common in many tumours. Patient-derived and healthy-donor monocyte-derived DC were examined for disruptions in mitogen-activated protein kinase (MAPK) signalling pathways associated with their capacity to generate Type-1 helper-T cell populations (Th1). Melanoma-lysate markedly suppressed TLR4-dependent IL-12p40 and p70 production. Suppression of IL-12p70 occurred independently of maturation markers (e.g., CD40, CD80, CD83) and correlated with depressed p35 and p40 transcription. Decreased IL-12 secretion was not associated with IL-10, TGFbeta, VEGF, PGE(2) or ganglioside in tumour lysates or attributable to endogenous PGE(2) release from DC. In contrast to HUVEC lysate, melanoma-lysate-treated DC were less able to generate Th1-responses from naïve T-cells. The p44/42 MAPK was hyper-activated by melanoma lysate, but not that of HUVEC. Blockade of MEK1/2, the upstream kinase for p44/42, with U0126 prevented p44/42 activation, restored IL-12p70, and permitted effective generation of Th1-responses. Therefore the p44/42 MAPK is a target for tumour-mediated immune suppression of DC resulting in transcriptional down-regulation of IL-12 p35 and p40 genes, reduced IL-12 secretion and suppressed Th1-responses. Pharmacological intervention in the MEK-p44/42 axis may be applicable to render DC resistant to the suppressive effects of tumour lysates and may form part of a combination immunotherapy.

Topics: Blotting, Western; Butadienes; Cell Line, Tumor; Dendritic Cells; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Immunotherapy, Adoptive; Interleukin-12; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Necrosis; Nitriles; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Th1 Cells; Transcription, Genetic

Esophageal adenocarcinoma often arises from Barrett's esophagus. Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) play critical roles in cell survival. We hypothesized that inhibition of these pathways in Barrett's adenocarcinoma would decrease cell proliferation and alter apoptosis in vitro.. Two Barrett's-associated adenocarcinoma cell lines, SEG-1 (wild-type p53) and BIC-1 (mutant p53), were treated with MAPK (U0126) and PI3K (LY294002) inhibitors at 20 microm concentrations. After 24 and 72 h, cell viability was measured by MTT assay. Apoptosis and necrosis were evaluated by the Annexin V-FITC assay. Statistical analysis was performed by ANOVA.. LY294002 and U0126 treatment produced significant reductions (range 15.7 to 62.0%, P < 0.05) in cellular proliferation at both 24 and 72 h in the SEG-1 cells. BIC-1 cell viability was reduced (39.3 to 56.4%, P < 0.05) at 72 h. Both early and late apoptotic activity were significantly increased (P < 0.05) in the SEG-1 cells using both inhibitors. Necrosis was significantly reduced (P < 0.05) using both inhibitors. No changes in either early or late apoptosis or necrosis were observed in the BIC-1 cells.. Herein, we report significant antiproliferative effects against Barrett's adenocarcinoma by MAPK and PI3K inhibition in vitro. Pro-apoptotic mechanisms prevail in the wild-type p53 cells. Further investigation is warranted to advance the clinical treatment of this devastating disease.

Topics: Adenocarcinoma; Apoptosis; Barrett Esophagus; Butadienes; Cell Division; Cell Line, Tumor; Cell Survival; Chromones; Enzyme Inhibitors; Esophageal Neoplasms; Humans; Mitogen-Activated Protein Kinases; Morpholines; Necrosis; Nitriles; Phosphoinositide-3 Kinase Inhibitors

The extracellular-regulated kinases (ERK) modulate cell proliferation and survival in response to several different stimuli and are therefore important drug targets. ERKs are activated by the dual phosphorylation kinase MEK1 and MEK1 inhibitors PD98059, U0126 and CI-1040 are now widely used to inhibit ERKs in cell and animal studies. In an analysis of ERK functions in astrocytes we found that PD98059 (100microM) failed to inhibit ERK phosphorylation but U0126 (50microM) inhibited ERK phosphorylation to approximately 80%. Surprisingly, U0126 also caused profound depletion of ATP in glucose-deprived cells, leading to death by necrosis. Since glucose-deprived cells depend mainly on mitochondrial ATP-synthase for ATP production, we tested whether U0126 or PD184161, a derivative of CI-1040, might inhibit ATP synthase activity, using 143B(Rho0) cells (which lack a functional F0 subunit) to further parse this effect. We found that the F1F0ATPase activity extracted from U0126- or PD184161-treated parental 143B cells or astrocytes was indeed inhibited by >or=80% suggesting a covalent change in the enzyme. However, F1F0ATPase activity extracted from similarly treated 143B(Rho0) cells was spared. Because F1F0ATPase activity in isolated mitochondria was not inhibited directly, we propose that U0126 and PD184161 inhibit ATP-synthase via an indirect action on F0. The MEK1 inhibitors also induced necrosis of other glucose-deprived cell types including primary neurons at the same concentrations required for inhibition of ERK phosphorylation. Thus, the MEK1/ERK signalling pathway may modulate ATP synthase function, and its inhibition may cause cells unable to perform glycolysis to die by necrosis.

Topics: Adenosine Triphosphate; Aniline Compounds; Animals; Astrocytes; Benzamides; Butadienes; Cell Death; Glucose; Humans; MAP Kinase Kinase 1; Mice; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Necrosis; Neurons; Nitriles; Proton-Translocating ATPases; Sympathetic Nervous System; Tumor Cells, Cultured

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

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.

Topics: Amino Acid Chloromethyl Ketones; Antigens, CD; Apoptosis; Butadienes; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; fas Receptor; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydrogen-Ion Concentration; Imidazoles; Inflammation; Interferon-gamma; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Necrosis; NF-kappa B; Nitriles; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pyridines; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; T-Lymphocytes; Tumor Necrosis Factor-alpha

Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126.. Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining.. U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses.. These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered.

Topics: Annexin A5; Apoptosis; Butadienes; Cell Division; Cell Survival; Enzyme Inhibitors; Fluorescent Antibody Technique; Humans; Interleukin-6; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Necrosis; Nitriles; Plasmacytoma; Protein Serine-Threonine Kinases; Reproducibility of Results; Scattering, Radiation; Staining and Labeling; T-Lymphocytes; Tumor Cells, Cultured