Abstract
Dysregulation of the mitochondrial signaling pathway of apoptosis induction represents a major hurdle in tumor therapy. The objective of the presented work was to investigate the role of the intrinsic (mitochondrial) apoptotic pathway in the non-small lung cancer cell line NCI-H460 upon induction of apoptosis using the highly bioactive TRAIL derivative Db-scTRAIL. NCI-H460 cells were TRAIL sensitive but an only about 3 fold overexpression of Bcl-2 was sufficient to induce a highly TRAIL resistant phenotype, confirming that the mitochondrial pathway is crucial for TRAIL-induced apoptosis induction. TRAIL resistance was paralleled by a strong inhibition of caspase-8, -9 and -3 activities and blocked their full processing. Notably, especially the final cleavage steps of the initiator caspase-8 and the executioner caspase-3 were effectively blocked by Bcl-2 overexpression. Caspase-9 knockdown failed to protect NCI-H460 cells from TRAIL-induced cell death, suggesting a minor role of this initiator caspase in this apoptotic pathway. Rather, knockdown of the XIAP antagonist Smac resulted in enhanced caspase-3 degradation after stimulation of cells with TRAIL. Of note, downregulation of XIAP had only limited effects on TRAIL sensitivity of wild-type NCI-H460 cells, but resensitized Bcl-2 overexpressing cells for TRAIL-induced apoptosis. In particular, XIAP knockdown in combination with TRAIL allowed the final cleavage step of caspase-3 to generate the catalytically active p17 fragment, whose production was otherwise blocked in Bcl-2 overexpressing cells. Together, our data strongly suggest that XIAP-mediated inhibition of final caspase-3 processing is the last and major hurdle in TRAIL-induced apoptosis in NCI-H460 cells, which can be overcome by Smac in a Bcl-2 level dependent manner. Quantitative investigation of the XIAP/Smac interplay using a mathematical model approach corroborates our experimental data strengthening the suggested roles of XIAP and Smac as critical determinants for TRAIL sensitivity.
Highlights
Worldwide, lung cancer is the most common cause of cancer-related death in men and the third highest in women, being responsible for more than 1.5 million deaths in 2012 (World Cancer Report 2014, World Health Organization)
NCI-H460 cells have been described as type II cells in literature, representing cells where death receptor-induced apoptosis is dependent on the mitochondrial apoptotic pathway to amplify effector caspase activation [17]
To analyze the relevance of the mitochondrial pathway for efficient apoptotic signaling in response to the dimerized targeted Db-scTRAIL, a subline of NCI-H460 stably overexpressing a FLAG-tagged Bcl-2 molecule (NCI-H460/Bcl-2) was established (Fig 1A)
Summary
Lung cancer is the most common cause of cancer-related death in men and the third highest in women, being responsible for more than 1.5 million deaths in 2012 (World Cancer Report 2014, World Health Organization). At the center of the cellular apoptotic program is a cascade of proteases, the caspases, the activation of which results in apoptosis. Two main signaling pathways have been delineated to initiate the apoptotic program, called the extrinsic and the intrinsic pathway [4]. The extrinsic pathway is induced by activation of transmembrane receptors of the so called “death receptor” subgroup within the TNF receptor family which initiate apoptotic signals after binding their specific ligands. Activated death receptors recruit intracellular adapter molecules and form the death-inducing signaling complex (DISC) comprising procaspase-8/-10. These initiator caspases become subsequently cleaved and activated within the DISC. They in turn cleave and activate downstream caspases, i.e. they initiate the caspase cascade
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