Sensitization of melanoma cells for TRAIL-induced apoptosis by BMS-345541 correlates with altered phosphorylation and activation of Bax.
Resistance to TRAIL (TNF-related apoptosis-inducing ligand)- induced apoptosis limits its therapeutic use. Different strategies of TRAIL sensitization and a dependency on Bax have been reported, but common principles of TRAIL resistance and the way of Bax activation remained poorly understood. Applying a melanoma model of TRAIL-sensitive and -resistant cell lines, efficient sensitization for TRAIL-induced apoptosis is demonstrated by the kinase inhibitor BMS-345541 (N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-1,2-ethanediamine hydrochloride), which targets IκB (inhibitor of κB proteins) kinase β (IKKβ). This effect was completely abrogated by Bax knockout as well as by Bcl-2 overexpression, in accordance with a Bax dependency. Early loss of the mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspases) clearly indicated the activation of mitochondrial apoptosis pathways. Of note, BMS-345541 alone resulted in an early Bax activation, seen by conformational changes and by Bax translocation. The synergistic effects can be explained by Bid activation through TRAIL, which inhibits Bcl-2, and the activation of Bax through BMS-345541. The critical roles of XIAP (X-chromosome-linked inhibitor of apoptosis protein), Smac and Bid were clearly proven by overexpression and siRNA knockdown, respectively. The way of Bax activation by BMS-345541 was unraveled by establishing new assays for Bax activation. These showed reduction of the inactivating Bax phosphorylation at serine-184, while the activating Bax phosphorylation at threonine-167 was enhanced. Thus, modulation of Bax phosphorylation appeared as tightly related to TRAIL sensitivity/resistance in melanoma cells, and therapeutic strategies may be considered.
Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany.
A
A
Berger
S-A
SA
Quast
M
M
Plötz
A
A
Kammermeier
J
J
Eberle
D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology*; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D008928:Mitochondria / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009842:Oligopeptides / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011810:Quinoxalines / Q000494:pharmacology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
C471109:4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; D051023:BH3 Interacting Domain Death Agonist Protein; D007093:Imidazoles; D016328:NF-kappa B; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011810:Quinoxalines; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C532637:SMAC peptide; D053221:TNF-Related Apoptosis-Inducing Ligand; D051636:X-Linked Inhibitor of Apoptosis Protein; D051028:bcl-2-Associated X Protein; D051550:I-kappa B Kinase; D020169:Caspases