Abstract
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.
Highlights
In melanoma, we had previously shown functional activity of TRAIL receptors;[5] some melanoma cell lines showed permanent TRAIL resistance and others developed inducible resistance upon TRAIL treatment.[6]
By screening for strategies to sensitize melanoma cells for TRAIL, we identified the IKK inhibitor BMS345541, which had shown antitumor activity already in different tumor models.[19,20]
The kinase inhibitor BMS-345541 was evaluated for identifying strategies to overcome melanoma cell resistance to TRAIL-induced apoptosis
Summary
We had previously shown functional activity of TRAIL receptors;[5] some melanoma cell lines showed permanent TRAIL resistance and others developed inducible resistance upon TRAIL treatment.[6]. Mitochondrial apoptosis pathways are controlled by Bcl-2 proteins, enclosing antiapoptotic (e.g. Bcl-2 and Mcl-1), activation of nuclear factor-kB (NF-kB) was related to apoptosis resistance of cancer cells.[16] the kinase inhibitor BMS-345541 (N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-1,2-ethanediamine hydrochloride) has been developed as I-kB (inhibitor of kB proteins) kinase b (IKKb), which induces degradation of IkBa, the inhibitor of NF-kB.[17] The role of NF-kB in TRAIL sensitivity of melanoma cells remained in question.[6,18] By screening for strategies to sensitize melanoma cells for TRAIL, we identified the IKK inhibitor BMS345541, which had shown antitumor activity already in different tumor models.[19,20] We characterized the molecular mechanisms and found that BMS-345541 resulted in an early activation of Bax via its altered phosphorylation, whereas NF-kB may exert its effects only at later steps
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