Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells.
Okadaic acid (OA) is the major component of diarrhetic shell fish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. We investigated the signal transduction pathways involved in OA induced cell death in HeLa cells. OA induced cytotoxicity and apoptosis at IC50 of 100nM. OA treatment resulted in time dependent increase in reactive oxygen species and depleted intracellular glutathione levels. Loss of mitochondrial membrane permeability led to translocation of bax, cytochrome-c and AIF from mitochondria to cytosol. The cells under fluorescence microscope showed typical apoptotic morphology with condensed chromatin, and nuclear fragmentation. We investigated the mitochondrial-mediated caspase cascade. The time dependent activation and cleavage of of bax, caspases-8, 10, 9, 3 and 7 was observed in Western blot analysis. In addition to caspase-dependent pathway AIF mediated caspase-independent pathway was involved in OA mediated cell death. OA also caused time dependent inhibition of protein phosphatase 2A activity and phosphorylation of p38 and p42/44 MAP kinases. Inhibitor studies with Ac-DEVO-CHO and Z-VAD-FMK could not prevent the phosphorylation of p38 and p42/44 MAP kinases. Our experiments with caspase inhibitors Ac-DEVD-CHO, Z-IETD-FMK and Z-VAD-FMK inhibited capsase-3, 8 cleavages but did not prevent OA-induced apoptosis and DNA fragmentation. Similarly, pretreatment with cyclosporin-A and N-acetylcysteine could not prevent the DNA fragmentation. In summary, the results of our study show that OA induces multiple signal transduction pathways acting either independently or simultaneously leading to apoptosis.
Jayaraj
R
R
Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior 474002, India.
Gupta
Nimesh
N
Rao
P V Lakshmana
PV
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002273:Carcinogens / Q000633:toxicity*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019319:Okadaic Acid / Q000633:toxicity*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
D002273:Carcinogens; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D019319:Okadaic Acid; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase; D020928:Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D020169:Caspases; D005978:Glutathione