title: Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via, mitochondrial mediated caspase-dependent mechanism.
abstract: 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.
authors.affiliation: Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India.
authors.forename: Jayaraj
authors.initials: J
authors.lastname: Ravindran
authors.forename: Nimesh
authors.initials: N
authors.lastname: Gupta
authors.forename: Mona
authors.initials: M
authors.lastname: Agrawal
authors.forename: A S
authors.initials: AS
authors.lastname: Bala Bhaskar
authors.forename: P V
authors.initials: PV
authors.lastname: Lakshmana Rao
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053179:Caspase 7 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003524:Cyclosporins / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005978:Glutathione / Q000032:analysis; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D019319:Okadaic Acid / Q000633:toxicity*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020298:U937 Cells; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D000873:Anthracenes; D051033:Apoptosis Inducing Factor; D003524:Cyclosporins; D005419:Flavonoids; D007093:Imidazoles; D011725:Pyridines; D017382:Reactive Oxygen Species; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C432165:pyrazolanthrone; D019319:Okadaic Acid; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9; D005978:Glutathione; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D000111:Acetylcysteine