Abstract
Physiological roles of microsomal (iPLA2γ) and cytosolic (iPLA2β)Ca2+-independent phospholipase A2 were determined in two different epithelial cell models. R- and S-enantiomers of the iPLA2 inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA2γ and iPLA2β, respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0-5.0 μM) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA2β siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA2γ siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA2β and iPLA2γ siRNA on cell number and protein was also determined, and iPLA2β siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA2γ siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA2β, but not iPLA2γ, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA2β inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA2β inhibition results in greater decreases in cell growth and proliferation than iPLA2γ, identifies specific phospholipids whose expressions are differentially regulated by iPLA2β and iPLA2γ, and suggests novel roles for iPLA2β in cell growth.
Footnotes
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doi:10.1124/jpet.106.105650.
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This work was supported by a Georgia Cancer Coalition Distinguished Scholar Grant, a University of Georgia Junior Faculty Grant (B.S.C.), and a University of Georgia Fellowship and an American Foundation of Pharmaceutical Education Fellowship (B.P.).
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ABBREVIATIONS: PLA2, phospholipase A2; iPLA2 β, cytosolic Ca2+-independent phospholipase A2; BEL, bromoenol lactone [(E)-6-(bromoethylene)-3-(1-naphthaleny)-2H-tetrahydropyran-2-one]; iPLA2 γ, microsomal Ca2+-independent phospholipase A2; HEK, human embryonic kidney; iPLA2,' Ca2+-independent phospholipase A2; PI, propidium iodide; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PtdCho, phosphatidylcholine; MTT, 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; ESI-MS, electrospray ionization-mass spectrometry.
- Received April 3, 2006.
- Accepted June 7, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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