okadaic-acid and pyrazolanthrone

Several lines of evidence have revealed that phosphorylation of amyloid precursor protein (APP) at Thr668 is involved in the pathogenesis of Alzheimer's disease (AD). Okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used in AD research models to increase tau phosphorylation and induce neuronal death. We previously showed that OA increased levels of APP and induced accumulation of APP in axonal swellings. In this study, we found that in OA-treated neurons, phosphorylation of APP at Thr668 increased and accumulated in axonal swellings by c-jun N-terminal kinase (JNK), and not by Cdk5 or ERK/MAPK. These results suggest that JNK may be one of therapeutic targets for the treatment of AD. [BMB Reports 2016; 49(7): 376-381].

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Anthracenes; Blotting, Western; Cells, Cultured; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; Neurons; Okadaic Acid; Phosphorylation; Rats; Up-Regulation

Sphingolipid mediators such as ceramide are pleiotropic regulators of cellular growth, differentiation and apoptosis. We investigated the role of ceramide biosynthesis, metabolism and actions in term human cytotrophoblasts syncytialized over 7 days in culture. Intracellular C16 ceramide levels increased modestly after 3 days in culture, then declined. Ceramidase was present at particularly high levels in syncytialized trophoblasts; inhibition of ceramidase reduced the degree of cell fusion. Exposure to short chain C8 ceramide or aSMase enhanced secretion of the differentiation marker hCG without affecting fusion or cell viability. In contrast, pharmacological inhibition of ceramidase reduced the extent of fusion. Inhibition of the ceramide-responsive JNK and PP2A pathways did not abolish the effects of ceramide, and JNK phosphorylation was unresponsive to ceramide; however, ceramide significantly inhibited phosphorylation of Akt. This study suggests that changes in ceramide biosynthesis and metabolism play a differential role in the biochemical and morphological features of trophoblast differentiation.

Topics: Anthracenes; Antigens, Differentiation; Caspase 8; Cell Differentiation; Cell Fusion; Cells, Cultured; Ceramidases; Ceramides; Chorionic Gonadotropin; DNA-Binding Proteins; Enzyme Inhibitors; Female; Gene Expression; Giant Cells; Humans; Nuclear Proteins; Okadaic Acid; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Processing, Post-Translational; Pyrans; Sphingomyelin Phosphodiesterase; Spiro Compounds; Transcription Factors; Trophoblasts

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Anthracenes; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Caspase 3; Caspase 7; Caspase 9; Cell Line, Tumor; Cyclosporins; Cytochromes c; DNA Damage; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glutathione; Humans; Imidazoles; Immunoblotting; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mitochondria; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Pyridines; Reactive Oxygen Species; U937 Cells