The anti-apoptotic effect of leukotriene D4 involves the prevention of caspase 8 activation and Bid cleavage.
We have shown in a previous study that leukotriene D(4) (LTD(4)) signalling increases cell survival and proliferation in intestinal epithelial cells [Ohd, Wikström and Sjölander (2000) Gastroenterology 119, 1007-1018]. This is highly interesting since inflammatory conditions of the bowel are associated with an increased risk of developing colon cancer. The enzyme cyclo-oxygenase 2 (COX-2) is important in this context since it is up-regulated in colon cancer tissues and in tumour cell lines. Treatment with the COX-2-specific inhibitor N -(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide has been shown previously to cause apoptosis in intestinal epithelial cells. In the present study, we attempted to elucidate the underlying mechanisms and we can now show that a mitochondrial pathway is employed. Inhibition of COX-2 causes release of cytochrome c, as shown by both Western-blot and microscopy studies, and as with apoptosis, this is significantly decreased by LTD(4). Since previous studies showed increased Bcl-2 levels on LTD(4) stimulation, we further studied apoptotic regulation at the mitochondrial level. From this we could exclude the involvement of the anti-apoptotic protein Bcl-X(L) as well as its pro-apoptotic counterpart Bax, since they are not expressed. Furthermore, the activity of the pro-apoptotic protein Bad (Bcl-2/Bcl-X(L)-antagonist, causing cell death) was completely unaffected. However, inhibition of COX-2 caused cleavage of caspase 8 into a 41 kDa fragment associated with activation and caused the appearance of an activated 15 kDa fragment of Bid. This indicates that N -(2-cyclohexyloxy-4-nitrophenyl)methane sulphonamide-induced apoptosis is mediated by the activation of caspase 8, via generation of truncated Bid, and thereafter release of cytochrome c. Interestingly, LTD(4) not only reverses the effects induced by inhibition of COX-2 but also reduces the apoptotic potential by lowering the basal level of caspase 8 activation and truncated Bid generation.
Wikström
Katarina
K
Division of Experimental Pathology, Department of Laboratory Medicine, University Hospital Malmö, Lund University, SE-205 02 Malmö, Sweden.
Juhas
Maria
M
Sjölander
Anita
A
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D017998:Leukotriene D4 / Q000494:pharmacology*; D008565:Membrane Proteins; D008928:Mitochondria / Q000378:metabolism; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D051025:bcl-Associated Death Protein
C494819:BAD protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D003574:Cytochrome c Group; D007527:Isoenzymes; D008565:Membrane Proteins; D009578:Nitrobenzenes; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D051025:bcl-Associated Death Protein; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D017998:Leukotriene D4; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases