Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells.
The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.
von Haefen
Clarissa
C
Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Humboldt University of Berlin, 13125, Germany.
Wieder
Thomas
T
Gillissen
Bernd
B
Stärck
Lilian
L
Graupner
Vilma
V
Dörken
Bernd
B
Daniel
Peter T
PT
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002458:Cell Fractionation; D002518:Ceramides / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D015251:Epirubicin / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
D000590:Amino Acid Chloromethyl Ketones; D000903:Antibiotics, Antineoplastic; C494826:BAX protein, human; D061945:Caspase Inhibitors; D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D015251:Epirubicin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases