Anandamide-induced apoptosis in Chang liver cells involves ceramide and JNK/AP-1 pathway.
In the present study we demonstrate that anandamide, the most important endogenous cannabinoid, markedly induced apoptosis in Chang liver cells, an immortalized non-tumor cell line derived from normal liver tissue, while it induced only modest effects in a number of hepatoma cell lines. The apoptotic effect was reduced by methyl-beta-cyclodextrin, a membrane cholesterol depletor, suggesting an interaction between anandamide and the membrane microdomains named lipid rafts. Anandamide effects were mediated by the production of ceramide, as demonstrated by experiments performed with the sphingomyelinase inhibitor, desipramine, or with the sphingomyelinase activator, melittin. This conclusion was confirmed by the observation that exogenous C2-ceramide induced a remarkable apoptotic effect in the same cells. Anandamide-induced apoptosis in Chang liver cells involved oxidative stress and activation of p38/JNK pathway, which was accompanied by a remarkable increase in AP-1 DNA-binding activity. Moreover, the levels of both c-Jun and JunB, two components of the AP-1 complex, and those of FasL and Bim, two transcriptional targets of AP-1, also increased during anandamide treatment. In addition, anandamide increased the level of Bax and caused degradation of full-length Bid with the production of the active truncated form. These effects were accompanied by dissipation of mitochondrial transmembrane potential with the consequent activation of both caspase-3 and caspase-6. On the contrary, in hepatoma cells, anandamide did not induce apoptotic effects and it was not possible to observe any increase in p38/JNK pathway and AP-1 activity after drug treatment. Our results suggest that the induction of cell death in non-tumor Chang liver cells by anandamide was mediated by ceramide, JNK and AP-1 and was dependent on the activation of both the extrinsic and intrinsic pathways of apoptosis.
Giuliano
Michela
M
Dipartimento di Scienze Biochimiche, Università di Palermo, Policlinico, Palermo, Italy.
Calvaruso
Giuseppe
G
Pellerito
Ornella
O
Portanova
Patrizia
P
Carlisi
Daniela
D
Vento
Renza
R
Tesoriere
Giovanni
G
D000111:Acetylcysteine / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001095:Arachidonic Acids / Q000494:pharmacology*; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000096:biosynthesis* / Q000494:pharmacology; D003891:Desipramine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology; D053284:Polyunsaturated Alkamides; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D048069:Tumor Necrosis Factors / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D001095:Arachidonic Acids; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; D002518:Ceramides; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D016166:Free Radical Scavengers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D053284:Polyunsaturated Alkamides; D011518:Proto-Oncogene Proteins; D018808:Transcription Factor AP-1; D048069:Tumor Necrosis Factors; D048031:JNK Mitogen-Activated Protein Kinases; D020169:Caspases; D003891:Desipramine; C078814:anandamide; D000111:Acetylcysteine