Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2.
Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.
Fuentes
Lucía
L
Institute of Molecular Biology and Genetics, University of Valladolid School of Medicine, E-47005 Valladolid, Spain.
Pérez
Rebeca
R
Nieto
María L
ML
Balsinde
Jesús
J
Balboa
María A
MA
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry; D002794:Choline / Q000378:metabolism; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D008264:Macrophages; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000648:ultrastructure; D009281:Naphthalenes / Q000009:adverse effects / Q000494:pharmacology*; D000073718:Pancreatitis-Associated Proteins; D010586:Phagocytes; D010711:Phosphatidate Phosphatase / Q000037:antagonists & inhibitors*; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D011753:Pyrones / Q000009:adverse effects / Q000494:pharmacology*; D013601:T-Lymphocytes; D014280:Triglycerides / Q000378:metabolism
D017304:Annexin A5; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D009281:Naphthalenes; D000073718:Pancreatitis-Associated Proteins; D010743:Phospholipids; D011753:Pyrones; C000612141:REG3A protein, human; D014280:Triglycerides; D016718:Arachidonic Acid; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D011433:Propranolol; D011065:Poly(ADP-ribose) Polymerases; D010741:Phospholipases A; D054467:Phospholipases A2; D010711:Phosphatidate Phosphatase; C505407:CASP3 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D008274:Magnesium; D002794:Choline; D002118:Calcium